Nuclear DNA

Somatic mutations accumulate within cells from aged humans and model organisms (Moskalev et al., 2012). Other forms of DNA damage, such as chromosomal aneuploidies and copy number variations, have also been found associated with aging (Faggioli et al., 2012; Forsberg et al., 2012). Increased clonal mosaicism for large chromosomal anomalies has been also reported (Jacobs et al., 2012; Laurie et al., 2012). All of these forms of DNA alterations may affect essential genes and transcriptional pathways, resulting in dysfunctional cells that, if not eliminated by apoptosis or senescence, may jeopardize tissue and organismal homeostasis. This is especially relevant when DNA damage impacts the functional competence of stem cells, thus compromising their role in tissue renewal (Jones and Rando, 2011; Rossi et al., 2008) (see “Stem Cell Exhaustion”).
Causal evidence for the proposed links between lifelong increase in genomic damage and aging has arisen from studies in mice and humans, showing that deficiencies in DNA repair mechanisms cause accelerated aging in mice and underlie several human progeroid syndromes, such as Werner syndrome, Bloom syndrome, xeroderma pigmentosum, trichothiodystrophy, Cockayne syndrome, and Seckel syndrome (Gregg et al., 2012; Hoeijmakers, 2009; Murga et al., 2009). Moreover, transgenic mice overexpressing BubR1, a mitotic checkpoint component that ensures accurate segregation of chromosomes, exhibit an increased protection against aneuploidy and cancer, as well as extended healthy lifespan (Baker et al., 2013). The latter findings provide experimental evidence that artificial reinforcement of nuclear DNA repair mechanisms may delay aging.

Mitochondrial DNA

Mutations and deletions in aged mtDNA may also contribute to aging (Park and Larsson, 2011). mtDNA has been considered a major target for aging-associated somatic mutations due to the oxidative microenvironment of the mitochondria, the lack of protective histones in the mtDNA, and the limited efficiency of the mtDNA repair mechanisms compared to those of nuclear DNA (Linnane et al., 1989). The causal implication of mtDNA mutations in aging has been controversial because of the multiplicity of mitochondrial genomes, which allows for the coexistence of mutant and wild-type genomes within the same cell, a phenomenon that is referred to as “heteroplasmy.” However, single-cell analyses have revealed that, despite the low overall level of mtDNA mutations, the mutational load of individual aging cells becomes significant and may attain a state of homoplasmy in which one mutant genome prevails (Khrapko et al., 1999). Interestingly, contrary to previous expectations, most mtDNA mutations in adult or aged cells appear to be caused by replication errors early in life, rather than by oxidative damage. These mutations may undergo polyclonal expansion and cause respiratory chain dysfunction in different tissues (Ameur et al., 2011). Studies of accelerated aging in HIV-infected patients treated with antiretroviral drugs, which interfere with mtDNA replication, have supported the concept of clonal expansion of mtDNA mutations originated early in life (Payne et al., 2011).
The first evidence that mtDNA damage might be important for aging and age-related diseases derived from the identification of human multisystem disorders caused by mtDNA mutations that partially phenocopy aging (Wallace, 2005). Further causative evidence comes from studies on mice that are deficient in mitochondrial DNA polymerase γ. These mutant mice exhibit aspects of premature aging and reduced lifespan in association with the accumulation of random point mutations and deletions in mtDNA (Kujoth et al., 2005; Trifunovic et al., 2004; Vermulst et al., 2008). Cells from these mice show impaired mitochondrial function, but unexpectedly, this is not accompanied by increased ROS production (Edgar et al., 2009; Hiona et al., 2010). Moreover, stem cells from these progeroid mice are particularly sensitive to the accumulation of mtDNA mutations (Ahlqvist et al., 2012) (see “Stem Cell Exhaustion”). Future studies are necessary to determine whether genetic manipulations that decrease the load of mtDNA mutations are able to extend lifespan.

Nuclear Architecture

Defects in the nuclear lamina can also cause genome instability (Dechat et al., 2008). Nuclear lamins constitute the major components of the nuclear lamina and participate in genome maintenance by providing a scaffold for tethering chromatin and protein complexes that regulate genomic stability (Gonzalez-Suarez et al., 2009; Liu et al., 2005). The nuclear lamina attracted the attention of aging researchers after the discovery that mutations in genes encoding protein components of this structure, or factors affecting their maturation and dynamics, cause accelerated aging syndromes such as the Hutchinson-Gilford and the Néstor-Guillermo progeria syndromes (HGPS and NGPS, respectively) (Cabanillas et al., 2011; De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003). Alterations of the nuclear lamina and production of an aberrant prelamin A isoform called progerin have also been detected during normal human aging (Ragnauth et al., 2010; Scaffidi and Misteli, 2006). Telomere dysfunction also promotes progerin production in normal human fibroblasts upon prolonged in vitro culture, suggesting intimate links between telomere maintenance and progerin expression during normal aging (Cao et al., 2011). In addition to these age-associated changes in A-type lamins, lamin B1 levels decline during cell senescence, pointing to its utility as a biomarker of this process (Freund et al., 2012; Shimi et al., 2011).
Animal and cellular models have facilitated the identification of the stress pathways elicited by aberrations in the nuclear lamina characteristic of HGPS. These pathways include the activation of p53 (Varela et al., 2005), deregulation of the somatotrophic axis (Mariño et al., 2010), and attrition of adult stem cells (Espada et al., 2008; Scaffidi and Misteli, 2008). The causal relevance of nuclear lamina abnormalities in premature aging has been supported by the observation that decreasing prelamin A or progerin levels delays the onset of progeroid features and extends lifespan in mouse models of HGPS. This can be achieved by systemic injection of antisense oligonucleotides, farnesyltransferase inhibitors, or a combination of statins and aminobisphosphonates (Osorio et al., 2011; Varela et al., 2008; Yang et al., 2006). Restoration of the somatotrophic axis through hormonal treatments or inhibition of NF-κB signaling also extends lifespan in these progeroid mice (Mariño et al., 2010; Osorio et al., 2012). Moreover, a homologous recombination-based strategy has been developed to correct the LMNA mutations in induced pluripotent stem cells (iPSCs) derived from HGPS patients, opening an avenue toward future cell therapies (Liu et al., 2011b). Further studies are necessary to validate the idea that reinforcement of the nuclear architecture can delay normal aging.

Overview

There is extensive evidence that genomic damage accompanies aging and that its artificial induction can provoke aspects of accelerated aging. In the case of the machinery that ensures faithful chromosomal segregation, there is genetic evidence that its enhancement can extend longevity in mammals (Baker et al., 2013). Also, in the particular case of progerias associated with nuclear architecture defects, there is proof of principle for treatments that can delay premature aging. Similar avenues should be explored to find interventions that reinforce other aspects of nuclear and mitochondrial genome stability, such as DNA repair, that could have a positive impact on normal aging (telomeres constitute a particular case and are discussed separately).

Telomere Attrition

Accumulation of DNA damage with age appears to affect the genome near to randomly, but there are some chromosomal regions, such as telomeres, that are particularly susceptible to age-related deterioration (Blackburn et al., 2006) (Figure 2A). Replicative DNA polymerases lack the capacity to replicate completely the terminal ends of linear DNA molecules, a function that is proprietary of a specialized DNA polymerase known as telomerase. However, most mammalian somatic cells do not express telomerase, and this leads to the progressive and cumulative loss of telomere-protective sequences from chromosome ends. Telomere exhaustion explains the limited proliferative capacity of some types of in-vitro-cultured cells, the so-called replicative senescence, or Hayflick limit (Hayflick and Moorhead, 1961; Olovnikov, 1996). Indeed, ectopic expression of telomerase is sufficient to confer immortality to otherwise mortal cells without causing oncogenic transformation (Bodnar et al., 1998). Importantly, telomere shortening is also observed during normal aging both in human and in mice (Blasco, 2007a).
Telomeres are bound by a characteristic multiprotein complex known as shelterin (Palm and de Lange, 2008). A main function of this complex is to prevent the access of DNA repair proteins to the telomeres. Otherwise, telomeres would be “repaired” as DNA breaks leading to chromosome fusions. Due to their restricted DNA repair, DNA damage at telomeres is notably persistent and highly efficient in inducing senescence and/or apoptosis (Fumagalli et al., 2012; Hewitt et al., 2012).
Telomerase deficiency in humans is associated with premature development of diseases, such as pulmonary fibrosis, dyskeratosis congenita, and aplastic anemia, which involve the loss of the regenerative capacity of different tissues (Armanios and Blackburn, 2012). Telomere uncapping and rampant chromosome fusions can also result from deficiencies in shelterin components (Palm and de Lange, 2008). Shelterin mutations have been found in some cases of aplastic anemia and dyskeratosis congenita (Savage et al., 2008; Walne et al., 2008; Zhong et al., 2011). Various loss-of-function models for shelterin components are characterized by rapid decline of the regenerative capacity of tissues and accelerated aging, a phenomenon that occurs even in the presence of telomeres with a normal length (Martínez and Blasco, 2010).
Genetically modified animal models have established causal links between telomere loss, cellular senescence, and organismal aging. Thus, mice with shortened or lengthened telomeres exhibit decreased or increased lifespans, respectively (Armanios et al., 2009; Blasco et al., 1997; Herrera et al., 1999; Rudolph et al., 1999; Tomás-Loba et al., 2008). Recent evidence also indicates that aging can be reverted by telomerase activation. In particular, the premature aging of telomerase-deficient mice can be reverted when telomerase is genetically reactivated in these aged mice (Jaskelioff et al., 2011). Moreover, normal physiological aging can be delayed without increasing the incidence of cancer in adult wild-type mice by systemic viral transduction of telomerase (Bernardes de Jesus et al., 2012). In humans, recent meta-analyses have supported the existence of a strong relation between short telomeres and mortality risk, particularly at younger ages (Boonekamp et al., 2013).

Overview

Normal aging is accompanied by telomere attrition in mammals. Moreover, pathological telomere dysfunction accelerates aging in mice and humans, whereas experimental stimulation of telomerase can delay aging in mice, thus fulfilling all of the criteria for a hallmark of aging.

Epigenetic Alterations

A variety of epigenetic alterations affects all cells and tissues throughout life (Talens et al., 2012) (Figure 2B). Epigenetic changes involve alterations in DNA methylation patterns, posttranslational modification of histones, and chromatin remodeling. Increased histone H4K16 acetylation, H4K20 trimethylation, or H3K4 trimethylation, as well as decreased H3K9 methylation or H3K27 trimethylation, constitute age-associated epigenetic marks (Fraga and Esteller, 2007; Han and Brunet, 2012). The multiple enzymatic systems assuring the generation and maintenance of epigenetic patterns include DNA methyltransferases, histone acetylases, deacetylases, methylases, and demethylases, as well as protein complexes implicated in chromatin remodeling.

Histone Modifications

Histone methylation meets the criteria for a hallmark of aging in invertebrates. Deletion of components of histone methylation complexes (for H3K4 and for H3K27) extends longevity in nematodes and flies, respectively (Greer et al., 2010; Siebold et al., 2010). Moreover, inhibition of histone demethylases (for H3K27) in worms may extend lifespan by targeting components of key longevity routes such as the insulin/IGF-1 signaling pathway (Jin et al., 2011). It is not yet clear whether manipulations of histone-modifying enzymes can influence aging through purely epigenetic mechanisms, by impinging on DNA repair and genome stability, or through transcriptional alterations affecting metabolic or signaling pathways outside of the nucleus.
Members of the sirtuin family of NAD-dependent protein deacetylases and ADP ribosyltransferases have been studied extensively as potential anti-aging factors. Interest in this family of proteins in relation to aging stems from a series of studies in yeast, flies, and worms, which reported that the single sirtuin gene of these organisms, named Sir2, had a remarkable longevity activity (Guarente, 2011). Overexpression of Sir2 was first shown to extend replicative lifespan in Saccharomyces cerevisiae (Kaeberlein et al., 1999), and subsequent reports indicated that enhanced expression of the worm (sir-2.1) and fly (dSir2) orthologs could extend lifespan in both invertebrate model systems (Rogina and Helfand, 2004; Tissenbaum and Guarente, 2001). These findings have recently been called into question, however, with the report that the lifespan extension originally observed in the worm and fly studies was mostly due to confounding genetic background differences and not to the overexpression of sir-2.1 or dSir2, respectively (Burnett et al., 2011). In fact, careful reassessments indicate that overexpression of sir-2.1 only results in modest lifespan extension in C. elegans (Viswanathan and Guarente, 2011).
Regarding mammals, several of the seven mammalian sirtuin paralogs can ameliorate various aspects of aging in mice (Houtkooper et al., 2012; Sebastián et al., 2012). In particular, transgenic overexpression of mammalian SIRT1, which is the closest homolog to invertebrate Sir2, improves aspects of health during aging but does not increase longevity (Herranz et al., 2010). The mechanisms involved in the beneficial effects of SIRT1 are complex and interconnected, including improved genomic stability (Oberdoerffer et al., 2008; Wang et al., 2008) and enhanced metabolic efficiency (Nogueiras et al., 2012) (see “Deregulated Nutrient Sensing”). More compelling evidence for a sirtuin-mediated prolongevity role in mammals has been obtained for SIRT6, which regulates genomic stability, NF-κB signaling, and glucose homeostasis through histone H3K9 deacetylation (Kanfi et al., 2010; Kawahara et al., 2009; Zhong et al., 2010). Mutant mice that are deficient in SIRT6 exhibit accelerated aging (Mostoslavsky et al., 2006), whereas male transgenic mice overexpressing SIRT6 have a longer lifespan than control animals, associated with reduced serum IGF-1 and other indicators of IGF-1 signaling (Kanfi et al., 2012). Interestingly, the mitochondria-located sirtuin SIRT3 has been reported to mediate some of the beneficial effects of dietary restriction (DR) in longevity, though its effects are not due to histone modifications but, rather, due to the deacetylation of mitochondrial proteins (Someya et al., 2010). Very recently, overexpression of SIRT3 has been reported to improve the regenerative capacity of aged hematopoietic stem cells (Brown et al., 2013). Therefore, in mammals, at least three members of the sirtuin family—SIRT1, SIRT3 and SIRT6—contribute to healthy aging.

DNA Methylation

The relationship between DNA methylation and aging is complex. Early studies described an age-associated global hypomethylation, but subsequent analyses revealed that several loci, including those corresponding to various tumor suppressor genes and Polycomb target genes, actually become hypermethylated with age (Maegawa et al., 2010). Cells from patients and mice with progeroid syndromes exhibit DNA methylation patterns and histone modifications that largely recapitulate those found in normal aging (Osorio et al., 2010; Shumaker et al., 2006). All of these epigenetic defects or epimutations accumulated throughout life may specifically affect the behavior and functionality of stem cells (Pollina and Brunet, 2011) (see “Stem Cell Exhaustion”). Nevertheless, there is no direct experimental demonstration thus far that organismal lifespan can be extended by altering patterns of DNA methylation.

Chromatin Remodeling

DNA- and histone-modifying enzymes act in concert with key chromosomal proteins, such as the heterochromatin protein 1α (HP1α), and chromatin remodeling factors, such as Polycomb group proteins or the NuRD complex, whose levels are diminished in both normally and pathologically aged cells (Pegoraro et al., 2009; Pollina and Brunet, 2011). Along with the above discussed epigenetic modifications in histones and DNA methylation, alterations in these epigenetic factors determine changes in chromatin architecture, such as global heterochromatin loss and redistribution, which constitute characteristic features of aging (Oberdoerffer and Sinclair, 2007; Tsurumi and Li, 2012). The causal relevance of these chromatin alterations in aging is supported by the finding that flies with loss-of-function mutations in HP1α have a shortened lifespan, whereas overexpression of this heterochromatin protein extends longevity in flies and delays the muscular deterioration characteristic of old age (Larson et al., 2012).
Supporting the functional relevance of epigenetically mediated chromatin alterations in aging, there is a notable connection between heterochromatin formation at repeated DNA domains and chromosomal stability. In particular, heterochromatin assembly at pericentric regions requires trimethylation of histones H3K9 and H4K20, as well as HP1α binding, and is important for chromosomal stability (Schotta et al., 2004). Mammalian telomeric repeats are also enriched for these chromatin modifications, indicating that chromosome ends are assembled into heterochromatin domains (Blasco, 2007b; Gonzalo et al., 2006). Subtelomeric regions also show features of constitutive heterochromatin, including H3K9 and H4K20 trimethylation, HP1α binding, and DNA hypermethylation. Thus, epigenetic alterations can directly impinge on the regulation of telomere length, one of the hallmarks of aging.

Transcriptional Alterations

Aging is associated with an increase in transcriptional noise (Bahar et al., 2006) and an aberrant production and maturation of many mRNAs (Harries et al., 2011; Nicholas et al., 2010). Microarray-based comparisons of young and old tissues from several species have identified age-related transcriptional changes in genes encoding key components of inflammatory, mitochondrial, and lysosomal degradation pathways (de Magalhães et al., 2009). These aging-associated transcriptional signatures also affect noncoding RNAs, including a class of miRNAs (gero-miRs) that is associated with the aging process and influences lifespan by targeting components of longevity networks or by regulating stem cell behavior (Boulias and Horvitz, 2012; Toledano et al., 2012; Ugalde et al., 2011). Gain- and loss-of-function studies have confirmed the capacity of several miRNAs to modulate longevity in Drosophila melanogaster and C. elegans (Liu et al., 2012; Shen et al., 2012; Smith-Vikos and Slack, 2012).

Reversion of Epigenetic Changes

Unlike DNA mutations, epigenetic alterations are—at least theoretically—reversible, hence offering opportunities for the design of novel anti-aging treatments (Freije and López-Otín, 2012; Rando and Chang, 2012). Restoration of physiological H4 acetylation through administration of histone deacetylase inhibitors avoids the manifestation of age-associated memory impairment in mice (Peleg et al., 2010), indicating that reversion of epigenetic changes may have neuroprotective effects. Inhibitors of histone acetyltransferases also ameliorate the premature aging phenotypes of progeroid mice and extend their lifespan (Krishnan et al., 2011). Moreover, the recent discovery of transgenerational epigenetic inheritance of longevity in C. elegans suggests that manipulation of specific chromatin modifications in parents can induce an epigenetic memory of longevity in their descendants (Greer et al., 2011). Conceptually similar to histone acetyltransferase inhibitors, histone deacetylase activators may conceivably promote longevity. Resveratrol has been extensively studied in relation to aging, and among its multiple mechanisms of action are the upregulation of SIRT1 activity, as well as other effects associated with energetic deficits (see “Mitochondrial Dysfunction”).

Overview

There are multiple lines of evidence suggesting that aging is accompanied by epigenetic changes and that epigenetic perturbations can provoke progeroid syndromes in model organisms. Furthermore, SIRT6 exemplifies an epigenetically relevant enzyme whose loss of function reduces longevity and whose gain of function extends longevity in mice (Kanfi et al., 2012; Mostoslavsky et al., 2006). Collectively, these works suggest that understanding and manipulating the epigenome holds promise for improving age-related pathologies and extending healthy lifespan.

Loss of Proteostasis

Aging and some aging-related diseases are linked to impaired protein homeostasis or proteostasis (Powers et al., 2009) (Figure 3). All cells take advantage of an array of quality control mechanisms to preserve the stability and functionality of their proteomes. Proteostasis involves mechanisms for the stabilization of correctly folded proteins—most prominently, the heat-shock family of proteins—and mechanisms for the degradation of proteins by the proteasome or the lysosome (Hartl et al., 2011; Koga et al., 2011; Mizushima et al., 2008). Moreover, there are regulators of age-related proteotoxicity, such as MOAG-4, that act through an alternative pathway distinct from molecular chaperones and proteases (van Ham et al., 2010). All of these systems function in a coordinated fashion to restore the structure of misfolded polypeptides or to remove and degrade them completely, thus preventing the accumulation of damaged components and assuring the continuous renewal of intracellular proteins. Accordingly, many studies have demonstrated that proteostasis is altered with aging (Koga et al., 2011). Additionally, chronic expression of unfolded, misfolded, or aggregated proteins contributes to the development of some age-related pathologies, such as Alzheimer’s disease, Parkinson’s disease, and cataracts (Powers et al., 2009).