Towards a unified mechanistic theory of aging

Experimental Gerontology 124 (2019) 110627

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Review

Towards a unified mechanistic theory of aging Gustavo Barja

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Department of Genetics, Physiology, and Microbiology, Faculty of Biological Sciences, Complutense University of Madrid (UCM), Spain

A R T I C LE I N FO

A B S T R A C T

Keywords: Aging Reactive oxygen species production Free radicals Longevity Autophagy

A large amount of the longevity-modulating genes discovered during the last two decades are highly conserved during evolution from yeast and invertebrates to mammals. Many different kinds of evidence converge in the concept that life extending manipulations like the dietary restrictions or rapamycin signal the nucleus specifically changing gene expression to increase longevity. The response of the cell aging regulation system is to change the level of activity of many different aging effectors to modulate longevity. Aging effectors include mitROS production, lipid unsaturation, autophagy, mitochondrial DNA repair and possibly others like apoptosis, proteostasis, or telomere shortening, corresponding to different classic theories of aging. The constitutive spontaneous activity of this aging regulating system, likely including epigenetics, can also explain species longevity. The aging regulating system reconciles the previously considered independent theories of aging bringing them together into a single unified theory of aging.

Section Editor: Christiaan Leeuwenburgh 1. Introduction. The mitochondrial ROS theory of aging as part of the cell aging regulating system Aging is the most significant risk factor for degenerative diseases (Bulterijs et al., 2015), including many forms of cancer, cardiovascular diseases, senile dementias and type-II diabetes, which cause around 40 million human deaths per year. Many factors are known to be involved in mammalian aging. This article brings together into a single unified theory the many previously considered independent “theories” of aging. In this new approach these do not correspond to “theories” but to different aging effectors (aging mechanisms). In this opinion article I expose my own views and theory concerning possible mechanisms of aging and their potential relationships to each other. The most robust environmental intervention that delays aging in most studied species, dietary (calorie) restriction (DR), increases longevity (Anderson and Weindruch, 2012) and/or delays degenerative diseases in mammals including rhesus monkeys (Colman et al., 2009; Mattison et al., 2012), although not all authors share this view (Hultström, 2015; Le Bourg, 2018). DR modifies levels of signaling

proteins like AMPK, mTOR (mammalian target of rapamycin) and many others in the cytoplasm of tissue cells, changing tissue-specific expression of hundreds of genes and > 20 biological functions (Fu et al., 2006), and changes RNA processing of genes associated with a highly integrated reprogramming of metabolism in rhesus monkeys (Rhoads et al., 2018). Weakness, slowness, poor endurance, low physical activity and frailty are also lower in DR than in AL-fed monkeys (Yamada et al., 2018). Rapamycin, a drug that increases mean and maximum longevity in a mammalian species (mice) over that of normal controls and increases healthspan (Harrison et al., 2009; Miller et al., 2014; Fischer et al., 2015; Bitto et al., 2016; Swindell, 2017), inhibits mTOR. The TOR pathway is involved in mitochondrial metabolism, ROS and lifespan determination (Schieke and Finkel, 2006). DR induces a specific lipidome and metabolome reprogramming in mouse tissues associated with lower levels of oxidative stress and protein oxidative damage (Jové et al., 2014). Metabolomic studies in rats of four different ages have identified 25 out of 120 metabolites that separate age classes strongly supporting two aging theories, those of energy dysregulation and free radicals (Son et al., 2012). DR increased survival in the Madison monkeys study (Colman et al., 2009), while the lack of improved survival in the Baltimore study

Abbreviations: AP, aging program; CARS, cell aging regulation system; DBI, double bond index; DR, dietary calorie restriction; DRs, dietary restrictions in general (referring to dietary, protein or methionine restriction); DSBs, double strand breaks; ETC, electron transport chain; 8-oxodG, (8-oxo-7,8-dihydro-2’deoxyguanosine); FRL, %free radical leak in the respiratory chain (mitROSp as % of total electron flow at the respiratory chain); MFRTA, mitochondrial oxygen free radical theory of aging; MetR, (isocaloric) methionine restriction; mitROSp, mitochondrial reactive oxygen species production; mTOR, mammalian target of rapamycin; nDNA, nuclear DNA; TF, transcription factor ⁎ Animal Physiology-II Unit, Faculty of Biological Sciences, Complutense University of Madrid (UCM), Street: c/Jose Antonio Novais n° 2, Madrid 28040, Spain. E-mail address: [email protected]. https://doi.org/10.1016/j.exger.2019.05.016 Received 5 March 2019; Received in revised form 8 May 2019; Accepted 30 May 2019 Available online 05 June 2019 0531-5565/ © 2019 Elsevier Inc. All rights reserved.

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Cybrids with haplogroups carrying the C150T transition have in common a lower mitROSp without changed respiratory capacity, mtDNA level, mitochondrial gene expression level, and growth rate (Chen et al., 2012). Mitochondria of long-lived animals secrete low levels of H2O2 to the cytosol. This can explain why long-lived animals have low levels of endogenous enzymatic and non-enzymatic antioxidants in total tissue (Barja et al., 1994a, 1994b; Barja, 2004a; PérezCampo et al., 1998) and why increasing total cellular antioxidants by dietary (Barja, 2004a) or genetic manipulations (Pérez et al., 2009a) does not modify longevity. Long-lived animals also have a low degree of fatty acid unsaturation in cell membranes (low DBI, double bond index), due to decreases in highly unsaturated fatty acids like 22:6n-3 and, depending on the species, to lower 20:4n-6 levels and higher levels of less unsaturated fatty acids like MUFAS or 18:2n-6 (Pamplona et al., 2002; Hulbert et al., 2007; Naudí et al., 2013; Cortie et al., 2015). Their lower DBI makes them highly resistant to lipid peroxidation in vivo (Pamplona and Barja, 2007). Most interestingly, manipulations that increase mammalian longevity, like dietary (caloric), protein, and methionine restriction and rapamycin treatment, also decrease mitROSp at complex I and oxidative damage to mtDNA (Gredilla et al., 2001; Barja, 2004a, 2004b; Sanz et al., 2006; López-Torres and Barja, 2008; Sánchez-Roman and Barja, 2013; Miwa et al., 2014) and the accumulation of mtDNA fragments inserted inside the chromosomal nuclear DNA (nDNA) during aging (Martínez-Cisuelo et al., 2016). However, it is not clear if differences in free radicals comparing species could also explain longevity differences between individuals (Stadtman, 2002). Moreover, oxidative damage can also have a signaling role (Radak et al., 2011). Mitochondria are not among the subcellular cell compartments with higher rates of ROS production. A much higher rate of ROS generation by NADPH oxidase occurs at the outer side of the plasma membrane of neutrophiles and other immune system cells, potentially causing strong damage in the extracellular fluids. Thus, ROS seem of paramount importance for aging both in the extra- and the intracellular compartments. Mitochondria, in spite of their relatively low well controlled and regulated rates of ROS generation, can be most relevant for aging because they are the only organelles, apart from the nucleus, that contain their own DNA. And their mtDNA can be easily damaged by the mitROS present on its vicinity, while nDNA is protected on a specially designed ROS- and lipid peroxidation-free nuclear environment. During the last decade it has been suggested that the mitochondrial ROS theory of aging is dead (Buffenstein et al., 2008; Pérez et al., 2009a; Sanz and Stefanatos, 2008). This was based on: a. the supposedly “oxidized state” of the very long-lived naked mole rats; b. the predominance of transitions vs. transversions in age-related mtDNA point mutations attributing them to replication errors instead of mitROSp; c. the lack of effect of the age related increases in mtDNA mutations and deletions due to their low levels and the high copy number and heteroplasmy of mtDNA; d. the lack of variation in mitROSp in mice mutant for mitochondrial pol γ (mtDNA polymerase γ); e. the hypothesis that ROS are not damaging to cells and instead act as signals. However these snags do not dismiss MFRTA because: a. the naked mole rats results apparently contradicting MFRTA were mainly based on unspecific measurements of oxidative stress (Andziak et al., 2006; Andziak and Buffenstein, 2006) and it was not stated if they were obtained in non-reproductive short-lived workers or in reproductive “queens”, the ones that live 30 years (reviewed in Barja, 2013); b. the low abundance of somatic G > T transversion mutations in mtDNA in tissues of aged animals cannot be used as an argument against MFRTA because it is caused by the nucleotide sensitivity of pol γ that prevents fixation of G > T transversions mutations even after abundant formation of 8-oxodG (8-oxo-7,8-dihydro-2’deoxyguanosine). The incorporated 8-oxodG causes > 95% replication blockade, but the remaining insertion of nucleotides occurs in the order dCTP ⋙ dATP > dGTP > dTTP, such as in the case of unmodified

(Mattison et al., 2012) could be due, among other reasons (Mattison and Colman, 2017), to the use of diets without precisely controlled composition, or to the fact that control “AL (ad libitum), fully fed” Baltimore monkeys ingested almost the same amount of calories per day as the DR Madison monkeys. Restricting the already restricted could help to explain the lack of positive effects on survival in the Baltimore study without abolishing the positive effects on the diseases. Interestingly, moderate DR in humans results in some of the same metabolic and molecular adaptations that improve health and retard the accumulation of molecular damage in animal models of longevity, improves multiple metabolic and hormonal factors implicated in the pathogenesis of type 2 diabetes, cardiovascular diseases, and cancer, and lowers body temperature, metabolic rate and oxidative stress (Most et al., 2017; Fontana et al., 2018; Gilmore and Redman, 2018). However, most of these human studies were performed in countries with a high level of obesity, and concerns about potential “side effects” of DR in humans have been raised (Dirks and Leeuwenburgh, 2006). Restriction of particular dietary components like proteins, or methionine (MetR), without caloric restriction, also increases rodent longevity with a potency around half that of the DR effect (Richie Jr et al., 1994; Miller et al., 2005; Sun et al., 2009; Sánchez-Roman and Barja, 2013). In addition, many single gene mouse mutants show increased longevity often in association with changes in hormones like insulin/IGF-1 (Brown-Borg, 2016; Tain et al., 2017; Newell Stamper et al., 2018) that affect similar cytosolic signaling proteins to those involved in DR effects. These proteins, in turn, modify gene expression (Lee et al., 1999; Anderson and Weindruch, 2010; Barger et al., 2017) modulating specific protein synthesis. Gene expression during DRs (dietary restrictions) can be also modulated by cytosolic and mitochondrial (SIRT 3-5) sirtuins (Satterstrom et al., 2015) and perhaps also by miRNAs (micro-RNAs) and larger non-coding RNAs (Abraham et al., 2017). The changes in gene expression modify the activity of aging effectors, helping to explain the observed increases in longevity. Reactive oxygen species (ROS) are the only known substances endogenously and continuously produced by cells that have the capacity to break covalent bonds. Many kinds of evidence continue to support the mitochondrial oxygen free radical theory of aging (MFRTA; Barja, 2013; Shen et al., 2014; López-Lluch et al., 2015; Latorre-Pellicer et al., 2016; Martín-Fernández and Gredilla, 2018; Miwa et al., 2014; Yen et al., 2018; Picca et al., 2018; Zsurka et al., 2018) both between and within animal species (Table 1). Long-lived mammals and birds have species-specific low mitochondrial ROS production rates (mitROSp) at complex I (Ku et al., 1993; Barja et al., 1994a, 1994b; Barja, 1999, 2004a, 2004b; Barja and Herrero, 1998; Herrero and Barja, 1997a, 1998; Lambert et al., 2007; Csiszar et al., 2012). This is obtained with almost zero energetic cost (Barja, 2013) and is associated with low levels of mtDNA oxidative damage (Barja and Herrero, 2000). Interestingly, mtDNA haplogroups carrying the C150T transition in the D-loop (Salvioli et al., 2008), and a complex I genotype, are associated with Northern Italian and Japanese centenarians respectively, the last one conferring resistance to degenerative diseases (Tanaka et al., 2000). Table 1 Summary of oxidative stress-related parameters in long-lived species and dietary restricted or rapamycin-treated rodents. mitROSp DBI FRL 8-oxodG in mtDNA mtDNA fragments in nDNA

Low in long-lived species, DRs, and rapamycin Low in long-lived species and 80% MetR Low in especially long-lived species (birds; bats & H.s.?), DRs, and rapamycin Low in long-lived species, DRs Low in rapamycin-treated mice, high in aging

DRs = dietary, protein or methionine restriction at 40% level. FRL = percent free radical leak at the mitochondrial respiratory chain. 8-oxodG = marker of oxidative damage to mtDNA. DRs and rapamycin increase mean and maximum longevity in mammals and rapamycin does the same in mice. For references see text. 2

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dG. Due to this, the selectivity of pol γ allows it to cope with high 8oxodG levels in mtDNA making the 8-oxodG positions less mutagenic (Graziewicz et al., 2007; Zsurka et al., 2018) while other ROS-induced changes like mtDNA breaks can still induce aging; c. the apparent problem for MFRTA that mtDNA deletions are present in most aged tissues at levels not enough to decrease global mitochondrial and cell function is solved by the discovery that the mtDNA fragments migrate to the cell nucleus and insert into nDNA (Section 3) which makes snags “b and c” irrelevant; d. the lack of effect of pol γ mutations on mitROSp is not a problem for MFRTA because mitROSp occurs upstream (cause) of mtDNA damage (effect). Therefore, it is illogical to expect that modifying the effect would necessarily modify its own cause; and e. the idea that ROS are signals instead of damaging substances forgets that ROS continue to be the only substances purposely produced by the organism that have the capacity to break covalent bonds, while interorganelle signaling is performed by a plethora of cytosolic proteins. Hypothetical nanomolar cytosolic ROS “signals” would be totally erased by local bursts of μM to mM H2O2 secretion from nearby mitochondria, making such signaling unreliable. Interpreting ROS as prosurvival signals due to reactive adaptations to their toxicity like antioxidant gene inductions (Wei and Kenyon, 2016; Sanz and Stefanatos, 2008) have been considered unlikely claims and oversimplifications ignoring the large wealth of knowledge supporting the traditional view (Liochev, 2013). Those claims are self-contradictory because they start denying the capacity of antioxidants to delay aging, to finally propose that longevity increases precisely due to adaptive increases in antioxidants and other protective substances. In any case, if finally the mitochondrial ROS generation rate would not be among the main contributors to the aging rate the robust negative correlation between mitROSp and longevity (Barja, 1999, 2004a, 2013; Lambert et al., 2007) would be very difficult to explain. Many of the issues mentioned above concerning MFRTA have been already reviewed in depth (PérezCampo et al., 1998; Gredilla and Barja, 2005; Hulbert et al., 2007; López-Torres and Barja, 2008; Barja, 1999, 2004a, 2004b, 2013; Pamplona and Costantini, 2011; Sánchez-Roman and Barja, 2013; Naudí et al., 2013) and will not be detailed again here. A large amount of the longevity-modulating genes discovered during the last two decades are highly conserved during evolution from yeast and invertebrates to mammals. In addition, available data now converge in the concept that experimental manipulations that increase individual longevity, like the DRs or rapamycin treatments, signal the nucleus to specifically change gene expression to slow down the aging rate. This concerted nuclear response changes the level of activity of many different aging effectors to modulate longevity. In this article mitROSp is considered as just one among various different aging effectors including the membrane fatty acid unsaturation (DBI), autophagy, and others. I specially focus on these three aging effectors because more evidence about their contribution to explain intra- and/or interspecies longevity is already available. But all of them work together integrated within a single highly regulated system. This article brings together into a single unified theory the many previously considered independent “theories” of aging. In this new approach these do not correspond to “theories” but to the different effectors (executors) of the aging program (AP) lying in the nucleus of each cell (Section 4). Together with the afferent sensing signals arriving from the cytoplasm, the extracellular medium and ultimately from the environment, the AP and its efferent effectors (the executors of aging) add up to constitute the Cell Aging Regulation System (CARS). The CARS model system has many advantages including viewing the various previously called different “theories” of aging as parts of a single integrated system instead of being mutually excluding. The CARS brings together the different “theories” into a single unified theory of aging.

2. The mitochondrial (%) Free Radical Leak indicates that mitROSp can be regulated During electron transport in the mitochondrial respiratory chain most but not all electrons reach the end of the chain to tetravalently reduce oxygen to water. A few among them univalently reduce oxygen to superoxide radical and then to other ROS. It is commonly assumed without evidence that these mitROS are “by-products” of an unwanted side reaction of the respiratory chain due to some kind of hypothetic evolutionary imperfection. Free radicals are involved in many useful pathways including apoptosis, development, thyroxin synthesis, immune system activities, or aging (Barja, 1993; de Magalhães and Church, 2006). MitROSp is not a simple by-product of mitochondrial respiration because it is regulated independently of O2 consumption in different physiologic situations, tissues, and animal species (Barja, 2007). If mitROSp were a simple by-product of the respiratory chain MFRTA would be wrong, because the rate of mitROSp would be directly proportional to the rate of mitO2 consumption. Comparing many mammalian species, both the weight-specific O2 consumption and mitROSp decrease as body size and longevity increase. But too many individual species and various whole groups like birds, bats and primates live much longer than expected when compared with the rest of mammals of similar body size and aerobic metabolic rate. These animals do not follow the Pearl's rate of living rule but they do not contradict the MFRTA. The reason for this is that the percent of total electron flow in the mitochondrial respiratory chain directed to ROS generation, the FRL (% free radical leak), is not a constant in all species. The exceptional longevity of various birds compared with mammals of a similar body size can be explained in part by their lower mitochondrial FRL. A low FRL means that the rate of mitROSp per unit of O2 consumption is lower in these birds. Their mitochondrial respiratory chain is more efficient in avoiding univalent lateral electron leaks to O2 than that of mammals following Pearl's rule, and this further decreases their mitROSp. Lower FRL and mitROSp occur in many organs of pigeons (longevity 35 years) compared to rats (longevity 4 years) in spite of their similar body mass and weight specific aerobic metabolic rates (Barja et al., 1994b; Herrero and Barja, 1997a; Barja and Herrero, 1998; Lambert et al., 2010). The rate of ROSp and the FRL are also lower in heart mitochondria of canaries and parakeets (longevity 24 and 21 years) than in those of mice (longevity 4 years) again in spite of their similar body mass and rate of aerobic metabolism (Herrero and Barja, 1998). The mitochondria of all these birds from three different Classes are more efficient than those of the mammals in transporting electrons to the final acceptor at the end of the electron transport chain (ETC), allowing them to generate less mitROS per unit of electron flow and O2 consumption, the decreased mitROSp contributing to their superior longevity. This lowering of FRL is a most interesting way of slowing mitROSp, because it can decrease the rate of aging without the need to decrease the aerobic metabolic rate and thus the general level of activity. This allows living longer while maintaining a high mitO2 consumption necessary for flight. Low FRL values have been also observed in bats compared to a similarly sized mammals (Brunet Rossinni, 2004; Brown et al., 2009). Interestingly, the same has been observed during chronic adaptation to aerobic exercise in mammals, which allows them to increase mitO2 consumption while avoiding the occurrence of strongly damaging increases in mitROSp (Herrero and Barja, 1997b; Barja, 2007). In addition, the FRL also decreases almost instantaneously during sudden increases in animal activity (aerobic exercise bouts) due to: i) decreases in the reduction state of the ETC complexes containing the mitROS generators, secondarily to the large increase in electron flow along the chain, and ii) local lowering of the pO2 at mitochondria secondarily to the large increase in O2 consumption of these organelles during exercise (Barja, 2007). If mitROSp would increase in proportion to mitO2 consumption exercise would be highly damaging and unhealthy, and animal activity would be strongly limited or almost non-existent. 3

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It has been simultaneously demonstrated that mtDNA fragments accumulate inside nDNA with age (Fig. 1) both in yeast (Cheng and Ivessa, 2010) and in rat liver and brain (Caro et al., 2010), and that such accumulation causes damage and accelerates aging in yeast (Cheng and Ivessa, 2012). Mouse liver also accumulates mtDNA fragments inserted inside nDNA with age (Martínez-Cisuelo et al., 2016). The insertion of those fragments in nDNA is non-random but a regulated aging-related process (Martínez-Cisuelo et al., 2016), and tends to occur flanked by retro-transposable elements in regions with high A + T and high DNA curvature and into open chromatin regions (Tsuji et al., 2012). These fragments were visualized under the microscope, using fluorescent in situ hybridization techniques, heavily concentrated at the pericentromeric regions co-localized with MaSat (major satellites) in mice. This age-related accumulation can solve the problem of the low abundance of mtDNA deletions in most somatic tissues. Thus, a main cause of aging seems to localize at mitochondria, the rate of mitROSp. But concerning its damaging consequences during aging we should look at the nucleus: the mtDNA fragments insertion inside nDNA, at the chromosomes. It has been described that rapamycin increases mouse longevity (Harrison et al., 2009). Strikingly, dietary treatment with rapamycin during 7 weeks in middle-aged mice decreased lipofuscin (non-autophagocytosed materials increasing with age) and fully reversed both mitROSp and mtDNA fragments accumulation in nDNA back to young levels (Martínez-Cisuelo et al., 2016). This suggests a cause-effect relationship between these two parameters because ROS have a strong capacity to produce both single and DSBs in DNA. Rapamycin also greatly lowers mtDNA deletions and ETC deficient muscle fibres in aging mice (Bielas et al., 2018). Therefore, part of the increase in longevity of rapamycin treated mice can be due to: (i) a decrease in mitROSp; (ii) a decrease in the insertion of mtDNA fragments inside nDNA; and (iii) an increase in autophagy. Concerning the mechanisms of damage induced by mtDNA fragments accumulation at the nucleus various possibilities have been proposed (Barja, 2018). The pro-aging actions of the mtDNA fragments inserted at pericentromeric regions can be facilitated by specific transposable element-mediated transport from those regions to the structural genes, or their regulatory regions. Such transport took place at least during evolution. Although most of the mtDNA fragments are present at the pericentromeric regions, mtDNA sequences are also found dispersed all along the length of all or most chromosomes (Gaziev and Shaikhaev, 2010; Matsuo et al., 2005; Michalovova et al., 2013; Singh et al., 2017). Chromosome loss, telomere shortening, and perhaps chromosome rearrangements seem to occur during aging (Telenti et al., 2016; Macedo et al., 2018). The strong abundance of mtDNA fragments at pericentromeric regions suggests a plausible mechanism of cellular damage by interfering with the mitotic machinery. The mtDNA fragments insertion in the pericentromeric region (Fig. 1) can have detrimental effects by affecting the nearby situated centromere causing chromosome missegregation during mitosis. That DNA region binds the modified histone CEN H3 (Cen PA) which binds in turn to microtubules. Therefore chromatid separation during mitosis could be affected by the inserted mtDNA fragments, generating chromosome abnormalities like aneuploidy which can be lethal to cells due to chromosome loss. Since most aneuploidies are lethal to individuals they are expected to cause also heavy damage at the cell level. Most interestingly it has been recently found that aneuploidy increases with age in human dermal fibroblasts due to dysfunction of the mitotic machinery increasing chromosome missegregation (Macedo et al., 2018). And this abnormality is not due to random damage. Instead it is controlled by FOXM1 (Macedo et al., 2018), a member of FOXO family involved together with other TFs (transcription factors) in many changes in longevity controlled by the CARS. Like in the case of telomers, these processes are expected to be most important in mitotic cells. The presence of aneuploid cells means that not only cell death but also cell malfunction occurs in some cells of old individuals.

However, the opposite is true. Exercise increases mean mammalian longevity, in agreement the decrease in mitROSp in vivo during exercise. It does not increase maximum longevity in spite of the decrease in mitROSp likely because: i) such decrease is limited to heart and skeletal muscle, which show large increases in O2 consumption during exercise, but does not occur in most other organs including the brain; ii) exercise lasts only a few minutes, a very small proportion of the 24 h long metabolic rate. On the other hand, exercise also increases cytosolic ROS production and its effects are systemic (Radak et al., 2019). The presence of low FRL values in species with exceptional longevity, and in the same species during acute and chronic aerobic exercise, shows that the amount of electrons diverted to ROS generation is not a fixed percentage of total electron flow. The FRL and the mitROSp are genetically determined and well-regulated flexible parameters instead of being simple “by-products” of mitochondrial respiration. A further relevant example for aging is DR. When laboratory rodents are subjected to the life extending dietary manipulations DR, protein restriction, or MetR, their FRL and mitROSp decrease below that of the AL-fed animals. This adaptation is established in a maximum of seven weeks and lasts for years as long as the dietary restriction is maintained (Gredilla et al., 2001; Sanz et al., 2004, 2006). Therefore, the percent of total electron flow in the mitochondrial respiratory chain is not a fixed fraction of total electron flow in various physiological situations. The FRL can vary depending on the mitochondrial state, during aerobic exercise bouts, after chronic exercise training, during DR, or in species with exceptional longevities like birds, bats, and likely in primates including humans. The FRL is decreased both inter-and intra-specifically during longevity extension as a regulated phenomenon. 3. MtDNA fragments insertion in nuclear DNA. A new mitochondrial-driven aging mechanism? It is known that accumulation of mtDNA fragments in nDNA occurs with age shortening lifespan (Fig. 1; Caro et al., 2010; Cheng and Ivessa, 2010, 2012), and that this accumulation is reversed by rapamycin (Martínez-Cisuelo et al., 2016), a treatment that increases longevity in mice. The occurrence of large mtDNA deletions, which increase with age in mammalian tissues, has been proposed as one final detrimental effect causing aging. Since mtDNA is highly compacted, without introns, the large deletions detected in old tissues would lead to the lack of many genes coding for electron transport chain or mitochondrial ribosome subunits in a single mtDNA circle molecule. However it is now clear that, with the exception of a few tissues, the level of these deletions does not reach the threshold needed to be of deleterious functional consequences in old animals. The high level of mtDNA heteroplasmia (the presence of both wild type and mutated mtDNA inside a single cell), due to the presence of thousands of mitochondria per cell and various mtDNA copies per mitochondrion, strongly protects against direct functional effects of mtDNA mutations. Only if a large majority of mtDNA copies are mutated mitochondrial ATP production would be compromised. Cells essentially homoplasmic for deleted mtDNA are abundant (between 43 and 60%) in a few cerebral areas like substantia nigra in humans (Bender et al., 2006; Kraytsberg et al., 2006), but in the brain in general and in other vital tissues of old individuals their percentage is too low (< 2%) to cause decrements in tissue function. Double strand breaks (DSBs) are most toxic among the many forms of DNA damage (Han et al., 2008), can produce mtDNA deletions in mtDNA and could drive aging (White and Vijg, 2016). They can be induced by ionizing radiation or ROS. But they can also generate mtDNA fragments, the missing overlooked segments deleted from mtDNA. In agreement with earlier proposals (Richter, 1988) those fragments can escape from mitochondria at least trough the permeability transition pore, have been observed in rodent brain cytosolic fractions (Patrushev et al., 2004, 2006), are present in nDNA (Tsuzuki et al., 1983; Singh et al., 2017), and are more heavily oxidized than wild type mtDNA (Suter and Richter, 1999). 4

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Fig. 1. Insertion of mtDNA fragments inside nuclear DNA increases with age and decreases with rapamycin treatment. MitROSp at complex I (Cx I) generates oxidized bases, and also double strand breaks at mtDNA that increase mtDNA deletions and mtDNA fragments. These fragments escape from mitochondria to the cytosol and enter the nucleus, where they insert into nuclear DNA mainly at pericentromeric (PCEN) area. Some of these fragments exit the PCEN and distribute along all the chromosome length. The mtDNA fragment insertions increase with age in nDNA in yeast and in rat and mouse tissues, promote chronological aging in yeast, and can cause aging and mortality through chromosome rearrangements like aneuploidy (due to chromosome mis-segregation), genetic instability and cancer. Therefore, although mitROSp is a main effector of aging, the main final target irreversibly damaged can be the nucleus. Short-term treatment with rapamycin (a drug which increases longevity) in middle aged mice fully reversed both the increases in mitROSp and in mtDNA fragments inserted in hepatic nDNA back to young levels (for references see text).

hand, the longevity of individual animals of a given species has been successfully increased up to 1.4 fold in mammals, either in DR animals or in single gene longevity mutants. This constitutes the “small effect”. Many other biogerontology-related facts discovered during the last decades, coming both from the laboratory and from studies in the wild, increasingly support the notion that aging can possibly be genetically programmed (Libertini, 1988; Barja et al., 1994b; Barja, 2010; Skulachev, 1997; Bowles, 1998; Guarente and Kenyon, 2000; Kenyon, 2001; Bredesen, 2004; Longo et al., 2005; Goldsmith, 2014; de Cabo et al., 2014; Jones et al., 2014; Mitteldorf, 2016a), which continues development. Intermediate positions between proponents of programmed and non-programmed aging have also appeared (de Magalhães, 2012; Lenart and Bienertová-Vašků, 2017; Flatt and Partridge, 2018). To defeat aging the small effect is not enough, and the cause underlying the “big effect” must be also unveiled. The “small effect” is likely controlled by the same CARS controlling the “big effect”, although in the former case the CARS output should be less intense and/ or should include a smaller number of aging effectors. The mitROSp and mtDNA fragments mentioned in the previous sections do not work in isolation. Instead, they are some among various different CARS aging effectors. The integrated CARS is composed of three main parts (Fig. 2):

Pericentromeric areas also influence chromosome binding to the inside of the nuclear envelope favouring chromosome movement, rearrangements, and local recombination. MaSat, where the mtDNA insertions have been localized, is also involved in heterochromatin formation and sister chromatid cohesion (Guenatri and Bailly, 2004). Although most signals from mtDNA fragments were observed at the pericentromeric areas, they seem to exit them, likely assisted by flanking TEs, and move to other chromosome regions where they have been mapped reaching more than 400 kbp in length (Singh et al., 2017) affecting structural genes and gene regulatory genomic regions causing abnormalities, aging (Fig. 1) and cancer (Singh et al., 2017). The inserted mtDNA fragments have been observed to increase by four fold inside nDNA both in colorectal adenocarcinoma genomes and in the patient's blood, and its presence correlates with higher mortality (Srinivasainagendra et al., 2017). MtDNA fragments are found in the blood in various diseases as well as in normally aged individuals, has a high pro-inflammatory capability (Franceschi et al., 2018), and is an independent predictor of mortality in the elderly (Storci et al., 2018). Therefore, mtDNA fragments may also have a role in systemic aging. 4. The cell aging regulating system (CARS) Different species can have hugely different species-specific life spans -up to one million difference-meaning that longevity must be written in the genome of each species. The genetically determined character of aging agrees with one of the four Bernard Strehler's rules of aging, the endogenous origin of aging (Strehler, 1962). This constitutes the “big effect”: the huge inter-specific differences in longevity. On the other

A) Cytoplasmic Pre-nuclear Signaling (mostly signaling proteins) B) The nuclear genetic Aging Program (AP) C) Post-nuclear Aging Effectors (executors of aging).

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Fig. 2. Cell Aging Regulation System (CARS). The CARS model broadly integrates known mechanisms of cellular longevity control. Different kinds of dietary restrictions (DR, MetR and protein restriction) and rapamycin, signals coming from the environment, alter humoral, endocrine, and finally cytosolic signaling proteins like mTOR, AMPK, PI3K, AKT, and many others (part A, left of figure) whose effects are mediated in many cases through TFs like FOXOs, TEFB and others that regulate expression of nuclear AP genes (part B center). The AP output (solid arrows leaving the nucleus on the right of the figure), in turn, modifies the activity of the multiple Aging Effectors (Part C, right) including: (a) ROSp in mitochondria, b) fatty acid double bonds (measured as double bond index, DBI) that stimulate lipid peroxidation, c) Autophagy, and d) likely others like apoptosis, inflammaging, proteostasis or telomere attrition. There is emerging evidence that that epigenetics is also involved in CARS action. The integrated response of the CARS to environmental signals modulates the intra-specific aging rate. An overlapping AP with additional components and wider output activity, composed only of parts B + C in the scheme, could also determine inter-species longevity. In some cases environmental signals can also directly reach aging effectors bypassing the nucleus, as shown for DR and mitochondrial NADH/NAD+. TFs = Transcription factors. * = telomere shortening will mainly occur in mitotic tissues.

Aging research has clarified many important facts that support programmed aging and CARS:

The rate of aging of each species (“big effect”) depends on the rates of activities of the aging effectors which, in turn, are constitutively determined by the AP of each species. In addition, the AP can also react to environmental signaling modifying the activity of its aging effectors to change the rate of aging of the individual (“small effect”). The CARS should be present both in mitotic and post-mitotic tissue cells. Mitotic cells could harbour additional hypothetical aging effectors like telomere shortening, apoptosis, or stop of cell division. These are not expected to be highly relevant in cells that do not or very rarely divide, like the majority of those in skeletal muscle, heart, and neurons in brain and neural tissues. Their relevance would be mainly limited to mitotic tissues like the gut or the skin. If cells do not divide, the telomeres do not shorten. Although apoptosis is likely to be more active in mitotic tissues, some role for apoptosis in aging of mitotic tissues cannot be ruled out. Conversely, aging effectors active in post-mitotic cells, like mitROSp, DBI, and autophagy, are also present in mitotic ones. Their role in aging is then expected to be most general at organism level. Although both mitotic and postmitotic cells can contribute to longevity, the most important tissues for aging are the ones mainly composed of post-mitotic cells, skeletal muscle, heart, or brain neurons. Multiple lines of evidence support the existence of a nuclear-coded AP that genetically determines the wide range of longevities observed among various animal species. Some authors (Dong et al., 2016; Olshansky, 2016) have argued that that human longevity cannot be increased over the 122 years limit (Olshansky, 2016). However longevity has repeatedly varied up to one million fold during biological evolution, including the approximately 10-fold increase that occurred from the earliest primates to man. Gerontology and molecular biology may be able to mimic such capacity in the future. It has been proposed that master genes that control the AP exist (Barja, 2008) and that it should be possible to slow down aging by decreasing the aging effects of the AP master genes, yielding a much longer youthful life.

a) After the first single gene mutation increasing mouse longevity was discovered (Brown-Borg et al., 1996), many other single gene mutations increasing mouse longevity by up to 40% have been found, and now > 40 of them are known (Liu et al., 2005; Folgueras et al., 2018; Yanai et al., 2017). In most cases eliminating these genes increases longevity, whereas it rarely decreases it, implying that their normal function is to promote aging. Many such genes are highly conserved in evolution (Bishop and Guarente, 2007) since they are homologous in organisms as different as yeast, nematodes like C. elegans, insects like Droshophila, and many vertebrates including mammals and humans. Therefore aging seems to be a very old trait of eukaryotic life (Clark, 2004). The apparently lower fitness of some of those mutants disappeared and became superior when digestive tract bacterial colonization was prevented (Podshivalova et al., 2017). A large number of aging related-genes is known. Some of them also improve fertility, but others do not. Around half of them do not have another known function. Most of the AP genes are likely nuclear coded (part “B” in Fig. 2) and promote aging by increasing the output of its pro-aging effectors. The best known among pro-aging genes are those of the GH-insulin/IGF1-like signaling pathway. These genes are already present in unicellular protists like yeast. Therefore, their ancient function should be to control aging instead of regulation of blood glucose. Only after > 500 million years of evolution from single-cell ancestors, when multicellular animals with a vascular system emerged, the need to regulate blood glucose and pancreatic insulin appeared and these genes were then co-opted for that secondary pleiotropic function. b) The AP has been proposed to be mediated by hierarchically 6

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organized aging gene clusters that progressively modify the individual during aging (Barja, 2008), analogously to Hox gene clusters involved in the control of development. The AP-controlled aging continues development and maturation. Most long-lived animal models consist in the possession of lower levels of pro-aging factors: “less is more in longevity”. Long-lived animals are “diluted” animals in the sense that they have less damaging pro-aging factors rather than more anti-aging defensive traits. Although the AP uses both pro- and anti-aging effectors, the pro-aging ones are more numerous. Proaging effectors reviewed here include mitROSp, DBI-lipid peroxidation, apoptosis, telomere attrition, mtDNA fragments inside nDNA, inflammation, or epigenetics, whereas anti-aging effectors of long-lived species are limited to autophagy. This is one reason why deleting longevity-related genes more frequently leads to increases than to decreases in longevity. The second reason is that signaling to the AP from the environment (e.g. nutrients) has also a pro-aging effect. That is why deleting genes coding for proteins involved in such signaling also leads to longevity increases. c) Although DR (and MetR and rapamycin) can also control aging in part by directly modifying the aging effectors, they do it mainly bringing about changes in expression of a large number of nuclear genes as shown by many microarray studies (Fu et al., 2006; Perrone et al., 2012; Plank et al., 2012; Pan and Finkel, 2017). The modified expression of those genes, in turn, modifies the activity of the aging effectors (Fig. 2). d) DR-induced changes in gene expression are species- and tissue-specific (Barger et al., 2017), are associated with energy metabolism, GH/insuling-like signaling, stress signaling, decreased carbohydrate and mitochondrial metabolism (Lee et al., 1999; Anderson and Weindruch, 2010), and also occur in rhesus monkeys (Rhoads et al., 2018). Together with the increase in longevity after inactivation of single genes codifying for pre-nuclear hormones, their receptors, cytoplasmic signaling proteins or transcription factors (part “A” of the CARS), this constitutes evidence that an AP exists in the cell nucleus. RNA-seq gene expression profiles in tissues of 33 mammals have uncovered parallel evolution of gene expression and lifespan, longevity variation explaining up to 18% variability in interspecies transcript levels, which is larger than intra-specific variation (Fushan et al., 2015). Thus, evolution of longevity seems to be due not only to changes in gene sequences but also to changes in gene expression. Evolution of longevity seems due to coordinated reprogramming of expression levels of many genes at the genome wide scale. This also supports the idea that inter-individual (“small effect”) and interspecies (“big effect”) differences in longevity can be under the control of different overlapping parts of a common nuclear AP. In the Fushan et al. (2015) study downregulation of gene expression was much more frequently observed than upregulation, agreeing with the observation that long-lived animals are mainly made up of less damaging (pro-aging) traits rather than of more protective (anti-aging) traits. Among protective factors, more repair of DNA DSBs (especially on primates) through non homologous end joining (NHEJ), and perhaps better telomere capping rather than length has been observed (Lorenzini et al., 2009). e) DR and MetR signal to tissue cells the abundance of food or proteins available for feeding in the external environment using humoral factors like insulin, GH/IGF-1, or blood amino acids like methionine. These in turn modify the activity of many cytoplasmic signaling proteins like IRS, PI3K, AKT, AMPK, sirtuins, mTOR complex, ERK, S6K, 4E-BP1, and many others (Johnson et al., 2013; Dibble and Cantley, 2015; Pan and Finkel, 2017). In the case of mTOR, we have found that rapamycin decreased mTOR downstream activity by lowering RAPTOR regulating protein instead of mTOR itself (Martínez-Cisuelo et al., 2016). Partial overlap among different afferent signals occurs, e.g. the existence of common set of genes induced by MetR and rapamycin (Azar et al., 2018). The intervention involving more afferent pathways is DR, in agreement with its

f)

g)

h)

i)

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higher induced increase in longevity compared to MetR, protein restriction or rapamycin. Many of the signals coming from the cytosolic longevity signaling proteins enter the nucleus, where the action of transcription factors like FOXOs, PPAR-α, PGC1-α, TFEB, ULK-1, SREBP, HIF-1, NRF-2, and many others (Fu et al., 2006; Hofmann et al., 2015; Martins et al., 2016) modify the expression of the AP genes involved in the control of longevity. Activation of FOXO TFs is linked to increases in longevity during DR. Functions induced by FOXO family TFs (Carter and Brunet, 2007) include DNA repair of DSBs, G2/M transition of mitotic cycle, increased autophagy (Lapierre et al., 2015), decreased mitROSp, and a decreased rate of cell aging (Raffaello and Rizzuto, 2011; Martins et al., 2016; Senchuk et al., 2018). Association of FOXO3 single nucleotide polymorphisms with human longevity in population studies has been also described (Willcox et al., 2016). However, such mechanisms have been criticized proposing that cellular processes do not modulate human aging as they do in nematodes or mice, due to their different life-histories (Le Bourg, 2016). The AP acts by varying the activity level of the post-nuclear aging effectors (Fig. 2C). Three of them have been better identified: (i) the mitochondria, and their rate of mitROSp at complex I, (ii) the degree of unsaturation of cell membrane fatty acids (DBI), and (iii) likely, the autophagy system. Concerning repair of endogenous nDNA damage, at variance with that corresponding to exogenous damage (Cortopassi and Wang, 1996; Barja, 2013), previous studies have found rather similar or less nuclear base excision repair enzyme activities both in DR and in long-lived compared to short-lived species (Stuart et al., 2004; Page and Stuart, 2011). However, it is unknown if mitochondrial base excision repair is higher in longlived species. If this were the case, in the case of mitochondria a long life would be achieved in part by combining both a low rate of damage generation (low mitROSp) and a high level of mtDNA repair. This could help to explain the strong negative correlation observed between mtDNA oxidative damage and mammalian longevity, not observed for nDNA (Barja and Herrero, 2000). Perhaps the same occurs for mitochondrial antioxidant enzymes. Indeed, the mitochondrial form of SOD, both MnSOD activity and protein level, was positively correlated with longevity in mammalian tissues and fibroblasts (Brown and Stuart, 2007; Page et al., 2010). In this line, the only antioxidant overexpressor mouse that has shown significantly increased maximum longevity was precisely the only one in which the antioxidant enzyme (catalase) was overexpressed inside the mitochondrial compartment (Schriner et al., 2005). It is then most important to investigate if the other mitochondrial antioxidant enzymes apart from MnSOD, contrarily to what happens for those present in total tissue (Pérez-Campo et al., 1998), also correlate positively with longevity, a most important overlooked possibility concerning MFRTA validity. Although a large part of the change in aging rate is controlled by the flow of information through the nuclear AP, the aging rate can be also changed via direct modification of mitROSp by dietary substances. This occurs in DR which decreases metabolites like pyruvate and others, lowering matrix NADH (low NADH/NAD+ ratio) and therefore decreasing complex I electronic reduction and mitROSp (Fig. 2 upper line). This could be a further mechanism by which nicotinamide nucleotide feeding in mice mitigates age-associated physiological decline specifically targeting mitochondria (Mills et al., 2016). Feeding humans with nicotinamide riboside increases mononuclear blood cell levels of NAD+ (Martens et al., 2018) lowering the NADH/NAD+ ratio. A similar effect through lowering of the NADH/NAD+ ratio is expected in other situations like exercise. Most long-lived single gene mutant mice live up to 40% longer (“small effect”) than the wild type animals. Interestingly, this coincides quantitatively with the maximum amount of longevity

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Three main such aging effectors, operative in aerobic vital tissues, that can contribute to increase longevity are decreased mitROSp at complex I, a decrease in cell membrane DBI, and increased autophagy. The contribution of the low mitROSp and DBI to long life is to decrease the rate of generation of (oxidative) damage at mitochondria and to have cellular membranes, including those of mitochondria, composed of fatty acid constituents that are more resistant to ROS-induced damage by having fewer double bonds (see Section 1). Autophagy removes cellular garbage. The three best known aging effectors can cause irreversible cellular damage through:

extension elicited by DR. This can perhaps occur because both correspond to the afferent arm of CARS (Fig. 2A) perhaps targeting similar and limited parts of the species AP, and thus similar aging effectors or total AP output activities. j) In addition to varying their expression in response to cytoplasmic, hormonal, and environmental signals, target and master AP genes can also be modified by epigenetic factors. Epigenetic changes like DNA methylation, and histone acetylation, methylation, phosphorylation, ubiquitination and sumoylation can be important factors in aging. During aging in general there is a decrease in global DNA methylation and an increase in local methylation at CpG islands and specific promoters (Jones, 2015; Johnson et al., 2012, Sun and Yi, 2015). Epigenetic marks establish changes in gene expression during cellular differentiation, and in response to environmental stimuli like DRs and rapamycin (D'Aquila et al., 2013). Some authors even support the existence of an “epigenetic clock” (Jones et al., 2015; Mitteldorf, 2016b; Horvath and Raj, 2018). This would be essentially different from the “epigenetic drift” during aging which corresponds to stochastic changes of small or not interest for the control of longevity (Jones et al., 2015). Selecting 353 CpG sites applying to different human tissues and cell types and to the entire human lifespan, it was observed that the “DNAm” (DNA methylation) age strongly correlated (r2 = 0.92) with chronological age (Horvath and Raj, 2018). This “epigenetic” clock thus emerges at least as a potential biomarker of aging (Horvath and Raj, 2018). Therefore, it is possible that epigenetics is also part of the AP (Fig. 2, part “B”).

1) An increase in mtDNA fragments insertion inside nDNA during aging induced by mitROSp. These fragments can shorten chronological life span because they have the potential to induce chromosome missegregation at mitosis leading to aneuploidies (Macedo et al., 2018), corrupt structural genes, stop cell division, modify regulatory sequences, and promote genomic instability. 2) Increased lipid peroxidation at cell membranes, especially at the strongly abundant mitochondrial ones (cristae) which are situated very close to the mitROS generator relevant for aging (Pamplona et al., 2004). Lipid peroxidation of cellular membranes is the strongest oxidative damage occurring inside cells due to the high intrinsic sensitivity to oxidative damage of the fatty acids containing a high number of double bonds. Lipid peroxidation is an exponential reaction chain process that generates many toxic and mutagenic lipid peroxidation products including short chain aldehydes like hydroxynonenal or malondialdehyde. The membrane lipid peroxidation derived aldehydes can diffuse throughout the cell including the nucleus, which is poor in lipids likely to avoid the dangerous lipid peroxidation process to occur on its interior where 99.99% of the genes and the rest of the genome are held. Reaching the nucleus by diffusion, those aldehydes chemically react with free amino groups in DNA and proteins. Malondialdehyde-dG adducts are present in nDNA (Chaudhary et al., 1994) and could contribute to DNA damage both in nDNA and mtDNA. In addition, lipid peroxidation products have the chemical reactive potential to generate crosslinked DNA and proteins that could make difficult gene expression since it needs to unpack DNA from the nucleosomes and unwind its twisted tangles. 3) Decreased autophagy that leads to the accumulation of heavily peroxidized and cross-linked lipids and proteins, decreased removal of injured and ROS-damaged mitochondria, and final accumulation of cellular garbage. The materials that cannot be digested and eliminated by autophagy accumulate in the cytosol as lipofuscin granules, the best known marker of aging at tissue level.

The CARS model integrates the main molecular mechanisms that are known to be involved in aging and longevity determination and is expected to be modified as new aging mechanisms are discovered. The term “hallmarks” of aging is frequently used (López-Otín et al., 2013; Folgueras et al., 2018; Partridge et al., 2018) to refer to different aspects of aging without any ordered causal organization between them. This also mixes primary aging effectors and their secondary derived damaging effects under a single term. This is solved using a model like CARS that minimizes confusion between afferent signals, causes, programs, aging effectors, and final damage forms linked to aging. These are different component parts of an overall system (CARS) that regulates cellular aging. The three main components of CARS, afferent signals, AP genes, and aging effectors (A-C in Fig. 2) work together, making possible both a species-specific basal aging rate and the longevity increase in response to DRs or rapamycin. In this last change the CARS modifies the aging rate in individuals of the same species (small effect). Between species (big effect) the CARS is composed only of parts “B” and “C” of Fig. 2, since its output activity is constitutive and species-specific and does not need any afferent signal stimulus to express itself. It is expected that parts of the AP are shared between and within species, although much higher output activity differences are expected in the case of the interspecies “big effect”. Partial overlap of CARS small and big effects is expected since at least mitROSp and complex I hydrophilic domain polypeptides, mTOR protein components, and fatty acid DBI vary both inside and between species in association with longevity (Barja, 2013; Gómez et al., 2014; Miwa et al., 2014). This shared use of AP components to control longevity inside and between species agrees with the general concept of the economy of structural gene numbers. Finally, the CARS mainly deals with the intracellular control of aging. Integration of the different CARS of different cells inside and between organs is needed to understand aging at whole organism level.

These three pro-aging processes occur at a lower rate in four known manipulations that increase longevity in mammals DR, PR, MetR, and rapamycin treatment: mitROSp is decreased, DBI is lowered, and autophagy is increased. In DR, DBI would be less relevant, in principle, than between species, because it only decreases at high (80%) and not at 40% MetR. Decreased insulin/IGF-1 and mTOR signaling also lower DBI (Admasu et al., 2018), like in long-lived mammals and birds compared to short-lived ones (Pamplona et al., 2002; Hulbert et al., 2007; Naudí et al., 2013). Autophagy has emerged as a third possible aging effector (Lapierre et al., 2015; Ren et al., 2017; Hansen et al., 2018), although it can also be maladaptive because it promotes cell death acting as a double edged sword. Autophagy is a cellular degradative process in which macromolecular aggregates and even whole organelles are degraded in lysosomes and the resulting materials are recycled to cell metabolism. Among the three types of autophagy, microautophagy, chaperonemediated autophagy, and macroautophagy (herein “autophagy”), the last one is the best understood. Autophagy is regulated by many signaling proteins including 15 autophagy-related (ATG) proteins, by

5. Three better known aging effectors In this article a main aging effector is defined as one fulfilling at least one of the following two requisites: a) it correlates with species longevities; and/or b) reacts to DR in individuals of a species; and does “a” or “b” in the appropriate predicted direction to increase longevity. 8

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neurons. If this were true, the importance of autophagy for aging would be further reinforced. A main key requisite for accepting any theory of aging is that it must be able to explain why different animals age at so different rates. The comparative approach can save a lot of research time and resources by easily discarding parameters not related to species longevity and can uncover further aging effectors additional to those represented on Fig. 2. Some CARS effectors, like mitROSp and DBI, have been studied using the comparative approach, and the results consistently showed lower mitROSp, lower oxidative damage to mtDNA, and lower DBI in long-lived species (Section 1). In the case of many other hypothetical aging effectors either comparative studies have not been performed or the results obtained did not support their involvement in aging. There is still no evidence that autophagy, apoptosis, proteostasis, telomere length, inflammation, mtDNA fragments inside nDNA, turnover-related parameters, or epigenetic markers correlate in the appropriate way with longevity across species. Concerning telomere attrition, among six different comparative studies, five found no correlation or negative correlation between telomere length or telomerase activity and longevity in tissues or fibroblasts of 7 to 60 species of mammals, and only one investigation reported positive correlation with longevity for the rate of telomere shortening in five birds (Stuart et al., 2013). Comparing values obtained in two different studies (Canela et al., 2007; Vera et al., 2012), it was estimated that mouse telomeres would shorten much faster than human telomers. Concerning proteostasis there is contradictory information since 20S and 26S proteasome, thioredoxin reductase, glutaredoxin activities, and ubiquitin levels showed no or negative correlation with mammalian longevity (Salway et al., 2011a), while in primates 20S but not 26S proteasomal activity was positively correlated with longevity (Pickering et al., 2015) and the very longlived naked mole rats showed superior protein stability and resistance to oxidative stress (Pérez et al., 2009b). In the same line, the specific protein carbonyls glutamic and aminoadipic semialdehydes and carboxymethyl- and carboxyethyl-lysine adducts in proteins (glycoxidation and lipoxidation protein markers) showed positive or no correlation with mammalian longevity while protein MDA-lysine adducts showed strong negative association with longevity (Ruiz et al., 2005). Concerning the relationship between turnover-related parameters like proteostasis and longevity there is the difficulty, in principle, that mammals of low body size like mice have higher weight-specific metabolic and protein turnover rates but have lower - instead of higher longevities that large-sized mammals. However the heat shock proteins mitochondrial HSP60, cytosolic HSP70 and endoplasmic reticulum GRP78 and 94 were positively correlated with mammalian longevity (Salway et al., 2011b). Many forms of cellular stress resistance, especially that to ROS-related factors including H2O2, organic peroxides, or paraquat, are also positively correlated with mammalian and bird longevity (Stuart et al., 2013). This can be related to the participation of those proteins in hormetic reactions, although hormesis, like exercise, seems to increase mean but not maximum longevity. Concerning DNA, DSB recognition and end-binding including the Ku80 and DNA-PKcs protein amounts are positively correlated with longevity in 13 mammals, the recognition varying by up to 100 fold between species (Lorenzini et al., 2009). Information on possible effects of DR on many candidates to aging effectors is also missing and should be investigated. Long-lived single gene mutant mice generally live up to 1.4 fold longer than their controls, similarly to what happens in DR. Most single gene mutation studies correspond to components on the sensitive (“afferent”) side of CARS (part “A” in Fig. 2). When the CARS components lying in the nucleus (part “B” in Fig. 2) are identified it is expected that increases in longevity much higher than 1.4 fold will be likely obtained, corresponding to the much larger range of lifespans that have evolved in different species. This will be especially possible through mutation or modification of the expression level of the master genes controlling the AP. Future studies should mainly focus on the aging effectors and,

mitochondrial phospholipids (Hsu and Shi, 2017) or by the DNA damage response (Eliopoulos et al., 2016) and involves three steps: initiation, nucleation and elongation. The autophagy concept as aging effector can be extended to any system with capacity to eliminate irreversibly accumulated final forms of macromolecular damage. Without enough “garbage cleaners” in relation to the rate of damage generation the progressive accumulation of cellular trash leads to impaired cell function as it seems to be the case with the heavy lipofuscin accumulation observed in some neurons. Heavy lipofuscin accumulation can exert damage at the cytosol and the mtDNA fragments damage the nucleus (Section 3), the concerted action of these two factors having the potential to damage the whole cell. Autophagy can eliminate heavily cross-linked, oxidized, and aggregated mixtures of peroxidized lipids and proteins (Linton et al., 2015) and even sometimes whole heavily damaged mitochondria (Biala et al., 2015), a process known as mitophagy (Green et al., 2011). Thus animal species age slowly if they have evolved to produce less toxic substances (ROS) per unit time, have membrane fatty acids that are more resistant to oxidation, and can better eliminate the remaining molecular damage by autophagy. This shows the strong interrelationship between these three main aging effectors. Many different mutant mice with single autophagy genes knocked out have a decreased life span, while DR increases autophagy in association with increased expression of autophagy controlling ATG genes induced by TFs like ULK1 or TFEB (Lapierre et al., 2015). Knocking out desaturase/elongase genes (which decreases DBI) increased longevity in C. elegans. On the other hand, mitROSp at complex I consistently decreases in all the four known experimental manipulations that increase mammalian longevity: DR, PR, MetR, and rapamycin feeding. A lower mitochondrial oxidative stress in the Ames dwarf long-lived mouse, which has low IGF-1 like signaling, and a higher one in shortlived GH transgenic mice have been also observed, in agreement with their respectively superior and inferior longevities (Brown-Borg et al., 2001; Sanz et al., 2002). In spite of some claims (Hansen et al., 2018) there is still no rigorous evidence that over-expression of autophagy genes increases mammalian longevity over that of normal controls, although mitophagy mediates lifespan extension under mitochondrial oxidative stress in C. elegans nematodes (Schiavi et al., 2015). Some evidence suggesting that increasing autophagy increases longevity has been published, although it was limited to a short-lived mouse strain in which overexpression of the essential autophagosomal protein ATG5 increased maximum lifespan from 781 to 900 days (Pyo et al., 2013). In contrast, in the first three experiments taken together in which rapamycin increased longevity the control mice of both sexes reached 1086 days and the rapamycin treated ones reached 1212 days of age at 90% mortality (Harrison et al., 2009), a longevity surpassed by DR. Husbandry conditions and the animal strain clearly affect the longevity attained in aging experiments. Experiments using longer lived control mice, approaching 1000–1200 days at least, are needed to identify other drugs that could increase longevity. During aging of postmitotic cells, such as neurons and cardiac myocytes, many mitochondria undergo enlargement and structural disorganization, while lysosomes, which normally contribute to mitochondrial turnover, gradually accumulate lipofuscin. These changes occur in part due to continuous mitochondrial oxidative stress and the inherent inability of cells to completely remove oxidatively damaged structures. Lipofuscin accumulation with age may also greatly diminish lysosomal degradative capacity by preventing lysosomal enzymes from targeting to functional autophagosomes, further limiting mitochondrial recycling. This interrelated mitochondrial and lysosomal damage irreversibly leads to functional decay and death of postmitotic cells (Terman et al., 2008). However there is debate concerning lipofuscin significance. While some have considered it only an excellent tissue aging marker, accumulation of lipofuscin could occupy large parts of cellular volume perturbing cellular functions as may occur in some 9

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senescent cells corrupt the interstitial fluid surrounding them strongly amplifying the damage by affecting neighboring cells, which can, in turn, also convert to additional senescent cells. The final result is multicellular amplification of damage by intercellular positive feedback loops that can contribute to aging and mortality. The detrimental effect of senescent cells for age-related decline was shown in studies demonstrating that clearing of p16INK4a-positive senescent cells from mice tissues using a novel transgene INK-ATTAC delayed the acquisition of age-related pathologies and phenotypes (Baker et al., 2011). Transplanting relatively small numbers of senescent cells from old to young individuals is sufficient to drive age-related conditions such as osteoarthritis, frailty, physical dysfunction and lowered survival in young mice (Xu et al., 2018). These advances raised a strong interest in trying to selectively kill senescent cells with senolytic agents, while leaving normal cells unharmed. Partial success has been already obtained with senolytic drugs like ABT-737 that targets the anti-apoptotic proteins BCL-W and BCL-XL (Yosef et al., 2016), or with combined quercetin plus dasatinib (Xu et al., 2018), that induce apoptosis and decreases the number of senescent cells lowering their secretion of proinflammatory agents, alleviating physical dysfunction and increasing late-life mice survival (Yosef et al., 2016). However, the selectivity of these agents for senescent cells was limited, it is known that quercetin does not increase mouse longevity (Spindler et al., 2013) and dasatinib is a chemotherapy agent showing significant toxicity. Using a modified version of FOXO4 TF, the senolytic FOXO4-DRI, induction of p53 nuclear exclusion induced apoptosis with greater selectivity eliminating up to 80% of senescent cells without affecting a significant fraction of normal cells, restoring fitness, fur density and renal function in naturally aging mice (Baar et al., 2017). After screening a panel of 10 flavonoids, fisetin was found as the most potent senolytic agent, reducing senescent cell burden in multiple tissues of aged wild-type mice and human explants, ameliorating blood clinical chemistry and histopathology, lowering lipid peroxidation and increasing GSH/GSSG ratio, and extending by around 12% median and maximum remaining lifespan of 85 weeks old animals, maximum longevity reaching around 135 weeks (2.6 years; Yousefzadeh et al., 2018). It is curious that many of the partially successful senolytics used are antioxidants, as the polyphenols quercetin or fisetin. Fisetin, at variance with some synthetic senolytics is a natural product present at low concentrations in many fruits and vegetables such as apples, onions, cucumbers, grapes and strawberries, has no reported adverse effects, and its average intake in Japan is around 0.4 mg/day. Interestingly, DR also prevents the accumulation of senescent cells in both mice and humans (Fontana et al., 2018), constituting a potential alternative approach to senolytic treatments without the need to increase apoptosis which could affect normal cells.

especially, on the nuclear AP. Most importantly, the interrelationship between the genes composing the gene clusters of aging and the master genes controlling them should be investigated. Available evidence favors the concept of a large multiplicity of aging effectors since none of them alone can explain the wide interspecies differences in longevity. Such multiplicity means that it is not logic to expect significant increases in longevity by varying a single aging effector or even a small part of a single one, as in the case of single antioxidant overexpressor mice concerning MFRTA. Therefore, the failure of single antioxidants to increase longevity (Buffenstein et al., 2008; Pérez et al., 2009a; Sanz and Stefanatos, 2008), does not necessarily indicate a lack of validity of a proposed aging effector. Although mitROSp varies between species, the magnitude of their differences in mitROSp is much smaller than their differences in longevity. Analogously, interspecies differences in autophagy or DBI, or the increase in longevity of long-lived single gene mutant mice are much smaller than the interspecies differences in longevity. Taken together, the action of the different aging effectors can perhaps explain such interspecies differences. The search and experimental confirmation of more additional aging effectors of CARS is needed. All these effectors should no longer be considered separate “theories of aging” but parts of an integrated system responsible for the control of longevity. 6. Three cell fates in aging tissues Aging effectors cause detrimental changes like genomic instability, lack of proteostasis, secondary inflammation, senescent cells, or stem cell exhaustion. The final result is irreversible cellular damage that can have three different fates: 1) Cell death by apoptosis or necrosis 2) Cell senescence and malfunction in postmitotic tissues 3) Malfunctioning cells in mitotic tissues which can lead to cancer There is evidence that during aging there is as a continuum of macro (chromosome) to micro (nucleotide) genomic changes in somatic cells expressed at the cellular and tissue levels. Prominent macro modifications include the loss of autosome and sex chromosomes, aneuploidies (Macedo et al., 2018) as well as less-documented abnormal chromosome rearrangements (Telenti et al., 2016) than can lead to genomic instability and then to cell malfunction and death through necrosis and apoptosis. On the other hand, many forms of cell damage including DNA damage, oxidative stress, aneuploidy, inappropriate autophagy, oncogenic mutations, metabolic and mitochondrial dysfunction, or telomere attrition, among others, can lead to cell senescence (Krtolica et al., 2001; White and Vijg, 2016; Macedo et al., 2018; Xu et al., 2018), a second kind of cell fate with worse detrimental effects for tissues than cell death. Cell senescence involves changes in cell morphology and activities, epigenetic modifications, extensive changes in gene expression including up-regulation of β-galactosidase, changed cellular activity and permanent cell cycle arrest induced by dysregulated p53-p21 or p16INK4a-pRB signaling. Increased activity of β-galactosidase staining, a common marker of cell senescence, is thought to be due to increased lysosomal mass. Senescent cells are scarce in the young but persist for long periods of time and accumulate with age in multiple tissues of old individuals of all mammalian species tested. The senescent cells develop a persistent pro-inflammatory phenotype called the senescenceassociated secretory phenotype (SASP) secreting to the surrounding tissue proinflammatory cytokines, chemokines, growth factors, matrix metalloproteinases, serine proteases, reactive oxygen (ROS) and nitrogen species, and extracellular matrix degrading proteins. Senescent cells can influence their surroundings through juxtacrine NOTCH/JAG1 signaling, intercellular communication, or exosome release, can reinforce their own senescence, induce senescence of neighboring cells (paracrine senescence), and have systemic effects. The malfunctioning

7. Conclusions The CARS model can explain modulation of longevity within species induced by environmental signals (DRs) or single gene mutations changing AP gene expression. A partially overlapping but larger AP could control species longevity. Between species the CARS is composed only by the AP and its aging effectors, since its output activity is constitutive and species-specific and does not need any afferent signal stimulus to express itself. More research is needed to better characterize CARS and its different aging effectors. Most aging research has focused on CARS afferent (pre-nuclear) signals or its post-nuclear aging effectors. To finally defeat aging, looking at the nucleus to identify the AP, especially its master genes, is needed. Further research is also needed to clarify if epigenetics is part of the AP and what are the main aging effectors acting at extracellular and systemic levels. Without the AP the different aging effectors and hallmarks of aging appear as independent separate entities that the cell aging regulating system reconciles and brings together into a single unified theory of aging. 10

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Funding

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