Model organisms have provided fundamental evidence that aging can be delayed and longevity extended. These findings gave rise to a new era in aging research aimed at elucidating the pathways and networks controlling this complex biological process. The identification of 9 hallmarks of aging has established a framework to evaluate the relative contribution of each hallmark and the interconnections among them. In this review, we revisit these hallmarks with the information obtained exclusively through the generation of genetically modified mouse models that have a significant impact on the aging process. We discuss within each hallmark those interventions that accelerate aging or that have been successful at increasing lifespan, with the final goal of identifying the most promising antiaging avenues based on the current knowledge provided by in vivo models.

Aging is becoming a major demographic phenomena of our time. However, although human life expectancy has remarkably increased worldwide in the past century, the length of time where individuals are in optimal health is much lower. Of note, aging is the main risk factor for the development of many pathological processes, such as cancer, cardiovascular disease, or neurodegenerative disorders, being age-related chronic diseases the most common cause of death.1 Therefore, extending healthy lifespan has become a major challenge for current and future society and the goal of geroscience.

The use of model organisms in aging research has been essential to achieve a key milestone in the field: the aging process can be modulated.2 Instead of just a passive, undefined decline of physiological functions, aging has turned out to be the result of a complex interconnection of genetic and biochemical mechanisms that have recently been categorized in 9 molecular hallmarks: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication.3 Although we are still far from understanding how this intricate network of pathways inexorably coordinates organismal deterioration, in vivo studies in animal models have proven that single genetic manipulations can extend lifespan or ameliorate certain age-related phenotypes. Also, external interventions, that is, caloric restriction, which target specific pathways, have demonstrated that aging can be delayed in a variety of species.

For several years, the use of invertebrate animal models—such as the worm Caenorhabditis elegans or the fruit-fly Drosophila melanogaster—has led aging research by providing the first insights into those molecular pathways that are determinant in the aging process and for lifespan extension. Despite the great progress achieved by using simple model organisms carrying mutations in specific genes, increasing efforts have been made during recent years to address whether these fundamental mechanisms are also shared by mammalian systems. In this regard, mouse models have become an excellent tool in aging research because of their relative short lifespan (which allows the monitoring of the aging process in a reasonable window frame) and to the feasibility of performing genetic manipulations. Also, mice share many of the age-related phenotypes found in human subjects, including the increased risk to develop certain diseases with age, such as cancer. Nevertheless, those age-related pathologies frequently found in elderly humans and absent in aged mice, such as certain cardiovascular (ie, atherosclerosis) or neurodegenerative (ie, Alzheimer) diseases, can be studied using the appropriate genetically modified mouse model already created to mimic these common human disorders.4 Accordingly, in this review, we revisit the hallmarks of aging through the prism of those biological insights provided exclusively by gain- and loss-of-function mouse models. We have focused on those genetic interventions that have a direct impact on a specific hallmark and discuss how this manipulation affects the aging process. Of note, the pleiotropic function of certain genes together with the inherent interconnection of some hallmarks makes sometimes difficult to point at a single molecular pathway/hallmark once a gene has been deleted or overexpressed. Finally, we have primarily highlighted those genetically engineered mice that shorten or increase healthy lifespan, keeping in mind that certain features of mouse models showing accelerated aging are not present in normal aging and vice versa.5

Genomic Instability

Throughout life, our DNA is exposed to a variety of exogenous and endogenous mutagenic agents, such as radiation, chemicals, or metabolic products, which cause different types of DNA damage, ranging from nucleotide modifications to single- and double-strand breaks. The inability to fully repair all DNA lesions makes inevitable the accumulation of somatic point mutations, deletions, integrations, or even chromosomal rearrangements in our genome over time, leading to increased genomic instability with age.6 Next-generation sequencing technologies have deepen into the classical paradigm that correlates DNA damage accumulation and aging. Despite this widely accepted association, causal evidence of the role of genome maintenance in the aging process has been provided by the existence of numerous human progeroid syndromes caused by mutations that jeopardize genomic stability. Accordingly, mouse models genetically engineered to mimic these human disorders also show DNA damage accumulation, accelerated aging phenotypes, and shortened lifespan. These mouse models can be categorized according to whether the genetic manipulation affects the integrity of nuclear DNA, mitochondrial DNA (mtDNA), or the nuclear architecture, which also causes genome instability (Figure 1).

Figure 1. Mouse models with shortened or extended lifespan generated by genetic manipulation of genes involved in genome maintenance. Green boxes indicate mouse models that show an increased longevity while red boxes indicate those with a reduced lifespan. Illustration Credit: Ben Smith. AAV9 indicates adeno-associated virus 9; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related; mtDNA, mitochondrial DNA; NER, nucleotide excision repair; and NHEJ, nonhomologous end joining.

Nuclear DNA

A large group of premature aging disorders have been found in humans and mice with mutations in components of the nucleotide excision repair (NER) machinery, one of the DNA repair systems that deals with single-strand lesions. Thus, mice double deficient in the NER components Csb (Ercc6) and Xpa show attenuated growth, cachexia, kyphosis, neurodegeneration, and shortened lifespan, mimicking the phenotype of patients with progeroid Cockayne syndrome.7 However, these accelerated aging features are not observed in mice where only Csb or Xpa has been removed and whose main phenotype is their high photosensitivity.8–10 This clinical feature is also found in patients with Cockayne syndrome and in those who experience Xeroderma pigmentosum, another disorder caused by mutations in core NER factors—such as XPA—and characterized by increased incidence of ultraviolet-induced skin cancer. Nevertheless, it is not always required to carry mutations in several components of the NER machinery to achieve age-related phenotypes in mice, other than cancer predisposition. Thus, genetically engineered mouse models deficient in Ercc1, Xpf (Ercc4), Xpg (Ercc5), or carrying a point mutation in Xpd (Ercc2) show premature aging features and reduced lifespan, which is not caused by increased tumor incidence.11–16 Instead, multiorgan dysfunction, including vascular aging, has been observed in many mouse models with severe genomic instability.17,18 Although impaired tissue functioning because of increased apoptosis or senescence has been proposed as one of the underlying mechanisms for the premature aging phenotype observed in these mice (further discussed below and in Cellular Senescence and Stem Cell Exhaustion sections), alterations in most of the molecular and cellular processes that constitute the hallmarks of aging have been reported for the progeroid models highlighted in this section.18 Of note, most of these mouse models deficient in NER components are characterized by growth retardation, a phenotype also found in human syndromes with mutations in these genes. Growth suppression and downregulation of the somatotroph signaling pathway have also been observed in many other progeroid disorders (see below), as well as in normal aging overtime. Interestingly, this feature has been postulated as a defense mechanism to counteract the aging process and extend longevity (see Deregulated Nutrient Sensing section).19,20

In addition to single-strand lesions, double-strand breaks are a dangerous source of genome instability. Thus, mice deficient in components of the nonhomologous end joining system involved in double-strand breaks repair and telomere maintenance, such as Ku80 (Xrcc5) and Ku70 (Xrcc6) and the catalytic subunit DNA-PKc (Prkdc), also display accelerated aging phenotypes.21,22 However, whereas Ku80-and Ku70-deficient mice show a dramatic reduction in lifespan without an increase in cancer incidence, the lack of DNA-PKc increased the frequency of lymphomas.21,22 Remarkably, another well-studied human progeria, such as the Werner syndrome, is caused by mutations in the RecQ DNA-helicase WRN (Werner), which also contributes to double-strand breaks repair and telomere homeostasis. In particular, the generation of mice deficient in Wrn emphasizes the relevance of telomeres in the aging process and the maintenance of genome stability (further discussed in section Telomere Attrition). Thus, Wrn−/− mice do not recapitulate human Werner syndrome clinical features unless mice are backcrossed for several generations to a background without the telomerase RNA component (Terc−/−) to shorten telomere length.23 It is important to highlight that mouse telomeres are much longer than human telomeres and therefore require more cell divisions to reach a critical length. Werner syndrome patients and Wrn−/−/Terc−/− mice develop their clinical symptoms once they reach the adulthood, therefore showing a slower onset than other progerias. Among these clinical features are reduced size, hair graying, alopecia, cataracts, osteoporosis, diabetes mellitus, and premature death caused by a higher incidence of cancer.23

Another key point in the maintenance of genome integrity is the accurate chromosomal segregation. Accordingly, mice hypomorphic for the spindle assembly protein BubR1 (Bub1bH/H) develop aneuploidy overtime and have shortened lifespan. However, contrary to what was expected, few mutant mice had tumors at the time of death. Instead, mutant mice show a variety of aging-associated phenotypes probably driven by the senescence state that has been observed in several tissues and that correlates with the degree of aneuploidy (further discussed in Cellular Senescence).24,25 Among them, the cardiovascular phenotype observed in Bub1bH/H mice, which includes reduced number of vascular smooth muscle cells, increased fibrosis, and arterial wall stiffening, has been considered as the main cause of death of these mice.25,26 Mutation in Bub1b has also been found in patients with Mosaic variegated aneuploidy syndrome, which has high aneuploidy rates, progeroid features, and increased cancer predisposition.27 Moreover, Bub1b overexpression in mice extends healthy lifespan. This mouse model is especially relevant because it provides causal evidence that improvement in DNA repair mechanisms increases longevity.28 Additional mouse models have been described with aberrant karyotypes, triggered by the failure of different mechanisms, which cause early aging-associated phenotypes. This is the case of mice double haploinsufficient for the mitotic checkpoint regulators Bub3 and Rae1 (Bub3+/−/Rae1+/−), mice hypomorphic for the replication stress regulatory protein Spartan (SprtnH/H), or mice deficient in the Cdc42 GTPase-activating protein (Arhgap1−/−) that regulates cytoskeleton organization and cell polarity.29–33

Overall, the generation of mouse models with mutations in the genes involved in the maintenance of genome integrity allows a fine unraveling of the roles that these factors play in the aging process, which sometimes are masked by the severity of certain human syndromes. As an example, the generation of a mouse carrying hypomorphic mutations in the DNA damage response protein ATR (Ataxia telangiectasia and Rad3-related), which triggers the Seckel syndrome in humans, has allowed the elucidation of novel functions for this protein in preventing age-related phenotypes.34 In agreement, conditional deletion of ATR in adult mice leads to a variety of premature aging features associated with a dramatic reduction in tissue renewal capacity caused by stem cell exhaustion.35 Likewise, because cancer predisposition frequently overwhelms the phenotype of many other human syndromes, which are also caused by defects in DNA repair mechanisms (Bloom syndrome, Fanconi anemia, Xeroderma pigmentosum, Ataxia telangiectasia, and Rothmund-Thomson syndrome, among others), new mouse models are needed to properly disentangle which type of DNA lesion triggers aging versus cancer phenotypes.36

Mitochondrial DNA

Similar to nuclear DNA, genetic material in the mitochondria is also susceptible to accumulate damage with age. Nevertheless, the coexistence of multiple copies of mtDNA per cell with a certain degree of variability among them, called heteroplasmy, raises the question of whether an increased rate of mutation overtime may truly compromise cell or tissue function.37 The generation of a mouse model carrying a mutation that decreases the proof reading activity of the mitochondrial DNA polymerase γ (PolgD257A/D257A or mtDNA mutator mice) provided causal evidence that a high load of mtDNA mutations triggered a variety of age-related phenotypes and shortened mice lifespan.38–40 Thus, mutant mice displayed kyphosis, alopecia, reduced bone mineral density and fat mass, anemia, sarcopenia, and an age-dependent cardiomyopathy characterized by a marked cardiac hypertrophy and fibrosis, among other clinical features.38–41 However, later studies highlighted the fact that the mutational load of PolgD257A/D257A mutant mice and the type of mtDNA mutations found in these mice were not comparable to the mutational spectrum accumulated in normal aging.42 Moreover, Polg+/D257A heterozygous mice, which exhibit a mutation frequency 500× higher than a 3-month-old age-matched wild-type mice and 29-fold higher than an old one (24–33 months), do not show any feature of accelerated aging or shorter lifespan.43 Nevertheless, regardless of its similarity to a normal aging context, this mouse model demonstrates that if mtDNA damage exceeds the mutation threshold that impairs mitochondrial function within a tissue, this dysfunction results in an age-related phenotype. Interestingly, studies with Polg mutant mice have also shown that low levels of heteroplasmic mtDNA mutations can be transmitted through the germline and that the combination of inherited and new somatic variants can exceed the mutation burden in postmitotic tissues, being enough to trigger age-related pathologies in the brain.44 Moreover, a recent work with conplastic mice (individuals with the same nuclear genome but different mtDNA variants) has demonstrated that mtDNA haplotype significantly influences the outcome of several age-related parameters and even results in lifespan differences among conplastic mice.45

Nuclear Architecture

Nuclear architecture defects can also lead to genome instability. In particular, nuclear lamins provide the scaffold needed for the maintenance of a myriad of nuclear protein complexes responsible for chromatin organization and gene regulation.46,47 Therefore, it is not surprising that mutations in lamins and lamin-binding proteins cause a variety of human diseases, called laminopathies, some of which unleash severe progeroid features, such as those found in the Hutchinson-Gilford progeria syndrome and in other disorders where the nuclear structure is compromised.48–51 The generation of mouse models mimicking the genetic defect found in some of these patients has provided further evidence that alterations in nuclear structural components cause age-related phenotypes. Thus, genetically engineered mice with mutations in the lamin A gene (LmnaG609G/G609G, LmnaL530P/L530P, LmnaHG) or deficient in the enzyme responsible for its accurate proteolytic processing and the conversion of prelamin A into the mature protein (Zmpste24−/−) accumulate post-translational modified unprocessed prelamin A or progerin (an aberrant prelamin A isoform) in the nucleus, leading to dramatic changes in nuclear morphology and function.52–55 The phenotype of most of these mouse models, similar to human patients, is characterized by shortened lifespan, growth retardation, alopecia, osteoporosis, kyphosis, lipodystrophy, and several cardiovascular pathologies, such as repolarization defects in the heart, as well as vascular calcification and loss of vascular smooth muscle cells and atherosclerosis (when progerin is ubiquitously expressed in Apoe−/− mice), ultimately leading to myocardial infarction.52–59 Remarkably, apolipoprotein E-null mice expressing progerin specifically in vascular smooth muscle cells exhibit all the cardiovascular alterations provoked by ubiquitous progerin expression and die prematurely.59 Interestingly, progeroid syndromes associated with nuclear architecture defects do not show neurodegeneration or increased susceptibility to cancer despite they also accumulate certain levels of DNA damage, in agreement with the key role of nuclear lamina in the maintenance of genomic stability.60

Further causal evidence for the role of nuclear architecture defects in the development of accelerated aging phenotypes has been provided by studies where different interventions aimed at decreasing the accumulation of post-translational modified prelamin A or progerin in the nuclear envelope, ameliorate several age-related features and extend the lifespan of Hutchinson-Gilford progeria syndrome mouse models.54,61,62 Moreover, the generation of a double mutant mice in which prelamin A methylation was reduced and mislocalized away from the nuclear rim (Zmpste24−/−/Icmthm/hm) completely prevented the premature death of these mice and the development of progeria symptoms.63

Telomere Attrition

Telomeres, the complex structures of hexanucleotide tandem repeats and bound proteins that protect the end of linear chromosomes, shorten with each cell division throughout life. Cell culture studies first demonstrated that critical telomere shortening triggered a DNA damage response that led the cells into a senescence status, called replicative senescence or Hayflick limit. This cell cycle arrest could be reverted by the ectopic expression of telomerase, the enzyme responsible for elongating eroded telomeres, which was sufficient to confer unlimited cell proliferative capacity or immortality. Telomerase is expressed in germ cells and adult stem cells, which are the ones that have the longest telomeres.64 However, this telomerase activity is not sufficient to prevent telomere attrition with age. The dependence of stem cell compartment on proper telomere fitness has been demonstrated by the finding that mutations in telomerase and telomeric proteins cause a variety of human diseases, called telomeropathies, which are characterized by the impairment of the self-renewal capacity of different tissues.65

The generation of genetically modified mouse models has been essential to provide experimental evidence of the cellular and organismal consequences of telomere shortening and its role in aging. Thus, characterization of mice lacking the telomerase RNA component of the holoenzyme (Terc−/−) revealed several age-related phenotypes, including alopecia, hair graying, reduced fertility, impaired wound healing and regenerative potential of hematopoietic and gastrointestinal systems, hypertension, and left ventricular dysfunction, as well as reduced lifespan.66–69 The detrimental effect of telomerase deficiency and of other telomeric proteins, such as Trf1 and Pot1b, in several stem cell compartments was further confirmed in subsequent studies using mice deficient in these proteins.70–74 Likewise, engineered mice with shorter telomeres and wild-type telomerase activity exhibit defects in the hematopoietic stem cell (HSC) compartment, which is primarily affected by telomere shortening.75 Additional evidence for the role of telomere length in aging has been provided by studies where the degenerative phenotypes of mice deficient in the catalytic subunit (Tert) of the telomerase complex can be rescued by genetic telomerase reactivation or by systemic viral transduction of Tert subunit.76,77 Moreover, treatment of old mice (1- or 2-year-old mice) with adeno-associated virus expressing Tert improved their healthspan and extended longevity in a normal aging context, without increasing cancer incidence.78 Of note, previous mouse models overexpressing Tert led to a cancer-prone phenotype (unless overexpressed in a cancer-resistant background),79 which is not surprising considering the ability of telomerase to confer an unlimited proliferative potential to cells. However, this recent study bypassed this handicap by expressing Tert temporarily because adeno-associated virus rarely integrate into the genome, thereby favoring its lost in rapidly dividing cells. Alternatively, chimeric mice containing cells with hyper-long telomeres and without increased telomerase activity have also been generated although its overall impact on aging and longevity remains to be elucidated.80

Epigenetic Alterations

A large body of experimental evidence has demonstrated that epigenetic patterns change throughout life. Noteworthy, enzymes responsible for histone modification, DNA methylation, or chromatin remodeling are sensitive to environmental changes. As an example, variations in cellular inputs, such as stress signals triggered by the aging process, can change the activity of certain transcription factors that are responsible for regulating the expression or recruitment of chromatin modifiers, therefore leading to epigenetic alterations overtime.81 To elucidate whether the observed changes in the epigenetic landscape with age are also a driving force behind the aging features, we can, once more, turn to the information provided by genetically modified model organisms. In this regard, much work has been done with invertebrates, where deletion or overexpression of chromatin modifiers has led to remarkable changes in lifespan extension.82 However, these studies have also revealed that the manipulation of the activity of 2 different enzymes that modify the same epigenetic mark may yield opposite results, suggesting that the effect observed on lifespan might be because of modifications performed on additional substrates targeted by these enzymes or to be context/loci dependent.81,82

Conversely, few mouse models based on genetic manipulations of histone-modifying enzymes have led to date to significant effects on lifespan. The existence of multiple paralogues in certain families of enzymes, in comparison to lower organisms, adds another level of complexity when evaluating the effect of single gene deficiency. The family of nicotinamide adenine dinucleotide–dependent histone deacetylases, named sirtuins (Sirt1–7 in mammals), stands among others as a result of early studies in invertebrates that conferred them a prominent antiaging role. In particular, overexpression of Sirt1, the closest paralogue to the gene Sir2, which was originally shown to extend longevity in lower organisms and now has been called into question,83 has demonstrated to ameliorate healthspan in mice, although not longevity, when globally overexpressed.84 Thus, it has been shown that Sirt1 contributes to telomere maintenance and genomic stability, as well as to improve metabolic functions in different tissues, which has been attributed to its deacetylase activity on nonhistone substrates.85–87 Remarkably, the only study that has reported an effect of Sirt1 on longevity has been achieved by specific Sirt1 overexpression in the brain (BRASTO mice), which drives hypothalamic neuronal activation and an intertissue communication that warrants further investigation.88 Likewise, whole-body Sirt6 overexpression extends lifespan in male, but not in female, transgenic mice, whereas Sirt6 deficiency causes age-related phenotypes. The effect of Sirt6 on longevity results from the combination of histone-modifying dependent and independent functions that ultimately regulate several metabolic pathways, genomic stability, nuclear factor (NF)-κB signaling, and stem cell maintenance.89–93 In addition, several studies have reported epigenetic-independent protective roles for the mitochondrial-located Sirt3 and Sirt5 in age-related pathologies, such as cardiac hypertrophy or Parkinson disease.94–96 The pleiotropy of sirtuin functions will also been discussed in Mitochondrial Dysfunction.

Alterations in the enzymes that modulate the patterns of histone methylation, another key epigenetic mark that may repress or activate genomic regions, have also led to significant effects on longevity in invertebrates.82 However, the role of these histone-modifying enzymes in mice has mainly been related to the maintenance of stem cell compartments, which, ultimately, may contribute to organismal fitness (see Stem Cell Exhaustion).97 In particular, mice deficient in Bmi1 (Bmi1−/−), a member of the Polycomb group that functions as an epigenetic repressor complex catalyzing the methylation of H3K27me3, show severe defects in the hematopoietic system and neurological abnormalities, which are associated with primary defects in HSC and neuronal stem cell self-renewal, in addition to other premature aging phenotypes.98 This phenotype has been linked to a derepression of senescent drivers, such as p16 and p19, as well as to an enhanced DNA damage response. In agreement, Bmi1−/− stem cells defects are markedly rescued by genetic deletion of the Ink4a/Arf locus or Chek2.99–101 Of note, conditional deficient mice in Mll1, a member of the Trithorax group that, opposite to Polycomb, catalyzes the methylation of the activating mark H3K4me3, also show defects in HSC self-renewal.102,103 This finding highlights the need of identifying which specific loci undergo changes in their epigenetic marks with age, rather than associating global alterations in the epigenetic patterns with the aging process.

Another line of evidence that provides a causal role for epigenetic alterations in the aging process comes from studies performed in mice with accelerated aging phenotypes. Thus, the progeroid mouse model Zmpste24−/−, which is characterized by profound nuclear alterations (as described in Genomic Instability), showed a marked hypoacetylation of H4K16ac that impaired the recruitment of DNA damage repair proteins. In agreement, treatment with a histone deacetylase inhibitor improved DNA repair, reduced cellular senescence, and extended Zmpste24−/− lifespan.104 Likewise, these mice also showed enhanced methylation of H3K9me3 caused by an aberrant stabilization of the methyltransferase Suv39h1, and whose genetic inactivation also improved lifespan of progeroid mice (double deficient Zmpste24−/−/Suv39h1−/−).105 Further evidence for the role of chromatin remodeling in the aging process has been provided by a recent and interesting study showing that partial in vivo reprogramming by short-term expression of the Yamanaka factors ameliorates age-associated hallmarks, both in an accelerated aging mouse model and in chronologically old mice.106 This study points to the reversion of some specific histone marks as responsible for the phenotype improvement, but given the complexity of the reprogramming process, further analysis of the epigenetic changes that underlie this phenomenon will be required.

Loss of Proteostasis

Many studies have demonstrated the existence of gradual accumulation of damaged/misfolded proteins and aggregates with age. In agreement, a great number of age-related pathologies, such as neurodegenerative diseases, are caused by a failure of the machinery responsible for the maintenance of protein homeostasis or proteostasis.107–109 This proteostasis network is mainly composed of chaperones, which assist protein folding and localization, and the proteasome and the lysosome-autophagy systems, which degrade those proteins that do not pass the quality control. The use of model organisms has provided causal evidence for the contribution of proteotoxicity to the development of many age-related diseases.110,111 Furthermore, manipulating the expression of the enzymes responsible for protein folding and degradation has significantly modulated the lifespan in invertebrates, indicating that an improvement of proteostasis may eventually delay the aging process.110,111

Regarding genetically modified mouse models, the number of studies that demonstrate an active role for proteostasis dysfunction in the aging process (excluding age-related diseases) is much more limited. Of note, the deficiency of certain key components of the proteostasis network leads to lethality in mice while the generation of tissue-specific mutants usually causes severe pathologies associated with a particular organ, hampering a global analysis of the role of proteostasis disruption in organismal ageing. Nevertheless, there are some remarkable studies that provide a causal link between protein homeostasis and healthy aging. Thus, hepatic overexpression of Lamp2 in old mice (Alb-Tet-off-L2A) is able to restore chaperone-mediated authophagy, which decreases with age, and to improve healthspan of aged mice.112 Likewise, deficiency of the cochaperone CHIP (carboxyl terminus of Hsp70-interacting protein; Stub1−/− mice) causes accelerated aging phenotypes, characterized by increased cellular senescence and oxidative stress, and a reduction in lifespan.113 More important, these mice show an accumulation of misfolded proteins that precedes most of the observed phenotypes, suggesting a causal role for proteostasis in the regulation of longevity.

Additional examples of lifespan modulation are found within the proteolytic systems. Thus, mice overexpressing a subunit of the proteasome that has a reduced chymotrypsin-like activity (Tg-β5t mice) accumulate polyubiquitinated and oxidized proteins and display shortened lifespan and premature aging phenotypes, such as lordokyphosis, reduced fat, atrophy, and degeneration of muscle fibers and increased senescence.114 Likewise, loss of the proteasome activator REGγ (11S regulatory particles, 28-kDa proteasome activator; Psme3−/− mice) accelerates aging although this phenotype has been directly linked to a specific impairment in the degradation of Mdm2 and the subsequent abnormal p53 accumulation.115 Conversely, enhanced protein clearance through the expression of a truncated form of the huntingtin protein (HttΔQ/ΔQ mice) that increases the level of autophagy extends lifespan in mice.116 Furthermore, whole-body overexpression of Atg5 (Tg-Atg5 mice), an essential protein for autophagy induction, led to a significant lifespan extension in mice and improved healthspan.117 In addition, tissue-specific deficient mouse models in other members of the Atg family, such as Atg7 and Atg12 (Atg7flox/flox:Vav-Cre, Atg7flox/flox:Pax7-Cre, Atg12flox/flox:Mx1-Cre), have demonstrated an essential role for autophagy in the maintenance of stem cell function.118–120

Additional interventions aimed at improving proteostasis (not based in genetic manipulations) have also extended lifespan in mice.121 However, it is not always clear the precise mechanism of action. As an example, rapamycin, a well-known inducer of autophagy, acts through inhibition of mammalian target of rapamycin (mTOR; mechanistic target of rapamycin) pathway, thereby decreasing protein synthesis and possibly affecting longevity by different means (see Deregulated Nutrient Sensing). Along these lines, a recent study has demonstrated that the reduced lifespan observed in worms and flies on CHIP inhibition is not because of a collapse of proteostasis but to an increase in the levels of insulin receptor (DAF-2), which is not further targeted for degradation.122 This study highlights the need of delving deeper into the relative contribution of global damaged/misfolded protein accumulation versus a deficient turnover of specific substrates relevant for the aging process.

Deregulated Nutrient Sensing

Dietary restriction was the first intervention able to extend lifespan in mammals.123–125 Several decades later, a Celegans mutant model showing the largest increased in longevity yet reported, confirmed the relevance of nutrient sensing pathways in the aging process.126 Since then, the insulin and insulin-like growth factor 1 (IGF-1) signaling pathway (IIS pathway), including upstream regulators (growth hormone [GH]) and downstream targets (mTOR), has been extensively modulated in model organisms, further demonstrating the essential role of nutrient and energy handling on longevity (Figure 2).

Figure 2. Genetic modulation of insulin and insulin-like growth factor 1 (IGF-1) signaling pathway (IIS) extends longevity in multiple mouse models. IIS pathway effectors are represented according to their physiological interactome. Green boxes indicate mouse models that show an increased longevity. Illustration Credit: Ben Smith. GH indicates growth hormone; IGFBP, IGF-binding proteins; mTOR, mechanistic target of rapamycin; and PTEN, phosphatase and tensin homolog.

Similar to the pioneer work with worms, the first study performed with mutant mice evidencing an extension in lifespan was performed with the Ames dwarf mouse (Prop1df/df mice), which has a point mutation in the Prop1 gene involved in somatotroph signaling.127 Prop1 activates the transcription factor Pou1f1 that is responsible for pituitary development and whose mutation also leads to increased longevity (Pou1f1dw/dw mice or Snell dwarf mice).128 Both mouse models show defective production of GH, thyroid-stimulating hormone, and prolactin, thereby showing severe growth retardation. However, the effects on longevity are primarily driven by the suppression of GH signaling.129 In agreement, mice deficient in the GH-releasing hormone (Ghrh−/−), with a point mutation in the GH-releasing hormone receptor (Ghrhrli/li or Little mice), or deficient in GH receptor (Ghr−/− or Laron mice) also show a remarkable extension in their lifespan compared with wild types (up to 50%).128,130,131 Consistent with the regulation of IIS pathway by GH signaling, all these mouse models showed a significant reduction in circulating insulin and IGF-1, as well as increased insulin sensitivity, which has been demonstrated to contribute to their slow-aging features.132

Additional strategies aimed at reducing insulin/IGF-1 signaling have also extended longevity in diverse genetically engineered mouse models. Thus, mice deficient in the pregnancy-associated plasma protein-A (Pappa−/−), a metalloproteinase that degrades IGF-binding proteins increasing IGF bioavailability, show a significant extension in longevity.133 Fat-specific insulin receptor–deficient mice (Insrflox/flox:aP2-Cre or FIRKO mice), insulin receptor substrate 1 knockout (Irs1−/−), and brain-specific insulin receptor substrate 2–deficient mice (Irs2flox/flox:Nes-Cre) also show increased lifespan.134–136 Nevertheless, the effect of these strategies on lifespan extension is lower than that obtained when GH signaling is compromised and, in some models, leads to insulin resistance. In addition, mice hypomorphic for IGF-1 exhibited only lifespan extension in females in 1 of 3 cohorts of mice bred in 3 independent institutions.137 Similarly, longevity studies with mice heterozygous for IGF-1receptor (Igf1r+/−) and mice deficient in Irs2 (Irs2−/−) yielded different results depending on the laboratory where the experiment was performed, suggesting that background and environmental differences play also a key role in lifespan extension.136,138–141 Along these lines, an interesting study showed that calorie restriction only increased lifespan in 10 of 41 mouse inbred strains analyzed, being detrimental in numerous cases.142 Of note, this report emphasizes the complexity of longevity studies and the influence of genetic background but also the great number of factors to take into account when designing this type of experiments.4

Decreasing the activity of the IIS pathway by modulating the expression of downstream effectors also improved the lifespan of several mouse models: hypomorphic for PI3K (phosphatidylinositol 3-kinase; Pik3caD933A/+), Pten-overexpressing (Tg-Pten), and Akt1 heterozygous (Akt1+/−).143–145 However, the phenotype reported for each line was different, indicating the existence of a wide variety of strategies to ameliorate the metabolic function and extend longevity. In this sense, mTOR stands as a promising target because it acts as a central regulator of both insulin signaling and anabolic metabolism. Thus, mTOR activity is regulated by IIS pathway, as well as by the levels of amino acids and the energy status. mTOR integrates those signals to promote anabolic processes, such as ribosomal biogenesis, protein synthesis, and autophagy.146 Inhibition of mTOR by rapamycin extends lifespan in various model organisms, including mice.147 In agreement, genetically modified mouse models hypomorphic for mTOR (MtorΔ/Δ), heterozygous for both Mtor and Mlst8 (Mtor+/−/Mlst8+/−), and deficient in the mTOR downstream effector S6K1 (Rps6kb1−/−) also show increased lifespan.148–150

Overall, all above-mentioned mouse models support the idea that decreased nutrient sensing and reduced anabolic metabolism promote longevity. Thus, recently characterized haploinsufficient myc mice (Myc+/−) illustrate this paradigm, showing low circulating IGF-1 levels, downregulation of mTOR signaling, and enhanced lifespan and healthspan although the mechanism of action is not yet known.151 Paradoxically, both long-lived and many progeroid mouse models show a suppression of the somatotroph axis, which has been postulated as a survival strategy against systemic damage aimed at decreasing growth in favor of somatic preservation and lifespan extension.152 However, exacerbation of this mechanism has also been demonstrated to be detrimental. Thus, progeroid Zmpste24−/−, which showed a dramatic reduction in IGF-1 levels, underwent a significant recovery on IGF-1 supplementation.153

Mitochondrial Dysfunction

A large body of evidence has demonstrated that mitochondrial function declines with age.154,155 In agreement, several progeroid mouse models exhibit mitochondrial defects, and some of their age-related phenotypes are rescued when improving mitochondrial homeostasis. Thus, mice deficient in telomerase show reduced mitochondrial biogenesis as a consequence of the p53 repression of PGC (peroxisome proliferator-activated receptor gamma coactivator)-1α and PGC-1β, 2 transcriptional coactivators essential for mitochondrial generation. p53 deficiency in a telomerase deficient background improves gluconeogenesis and doxorubicin-induced cardiomyopathy in these mice.156 Similarly, mitochondrial dysfunction has been observed in mouse models with defects in DNA repair, such as Xpa−/− or Ercc6−/−, where DNA damage activates PARP1 (poly-ADP-ribose polymerase 1) causing nicotinamide adenine dinucleotide consumption and reduced Sirt1 activity, which in turn decreases PGC-1α expression. Hence, treatment with nicotinamide adenine dinucleotide precursors enhanced Sirt1 activity and PGC-1α levels, thereby ameliorating mitochondrial phenotype of progeroid mice and extending lifespan in mutant worms.157,158 Likewise, nicotinamide adenine dinucleotide repletion improves mitochondrial function and delays stem cell senescence in physiological old mice, even leading to a slight increase in their longevity.159 In addition, as mentioned in Genome Instability, mtDNA mutator mice develop premature aging, further supporting a causal role for mitochondrial dysfunction in the aging process. Finally, stimulation of mitochondrial biogenesis through endurance exercise attenuated age-related features in these mice.160

The precise mechanism by which mitochondrial dysfunction contributes to the aging process still remains unclear. The free radical theory of aging attributes a major role to the oxidative damage caused by the increased production of reactive oxygen species (ROS) over time, triggered by defective mitochondrial function. In agreement, the accelerated aging phenotype observed in p62-deficient mice (Sqstm1−/−) has been linked to an impaired activation of the master antioxidant regulator Nrf2 although the hypothesis of inefficient mitophagy caused by p62 deficiency has not been evaluated.161 However, mtDNA mutator mice, which exhibit a dramatic mitochondrial dysfunction and premature aging, do not show increased ROS.38,39 Neither, the deficiency in the mitochondrial protein Cisd2, which causes Wolfram syndrome 2 in humans and fatal mitochondrial degeneration and premature aging in mice, correlates with a significant difference in ROS levels compared with wild-type animals.162 In addition, many genetically modified mice deficient in enzymes responsible for the antioxidant defense display normal lifespan despite having enhanced ROS levels.163 Nevertheless, several of these mouse models show an increased susceptibility to the development of age-related diseases.163 However, none of the transgenic mice overexpressing antioxidant enzymes that have been generated to date show increased longevity, with the exception of mice that overexpress human catalase in the mitochondria (mCAT mice) and show an extension in median and maximal lifespans, which has been partially attributed to an attenuation of cardiac aging in these mice.164,165 Moreover, mice with reduced expression of glutathione peroxidase 4 (Gpx4+/− mice) show a slight increase in their longevity.166 This surprising result agrees with other studies in invertebrates, suggesting that certain levels of ROS or mild mitochondrial damage trigger survival signals, leading to the so called hormetic response, which ultimately may improve cellular fitness and lifespan. In this regard, mice deficient in Surf1 (Surf1−/−), a cytochrome c oxidase assembly factor, as well as mice heterozygous for Clk1 (Clk1+/−), an enzyme involved in ubiquinone biosynthesis, live significantly longer than wild types.167,168 Interestingly, the mitochondrial perturbation caused by Surf1 deficiency produced a variety of mitochondrial compensatory responses in Surf1−/− mice, which included increased mitochondrial biogenesis, upregulation of the mitochondrial unfolded protein response, and induction of the master antioxidant regulator Nrf2.169 Similar observations have been made in worms deficient in Clk1.170 Therefore, it is still a major challenge to define the precise threshold for dysfunctional mitochondria to become beneficial or detrimental for the aging process.

Finally, a recent study has demonstrated that mitochondrial dysfunction can also induce senescence with a distinctive secretory profile, which has been named mitochondrial dysfunction-associated senescence.171 This type of senescence has been found in vivo in mtDNA mutator mice, adding another level of complexity to the role of mitochondria in the aging process that will require further investigation.

Cellular Senescence

Recent findings based on the use of genetically modified mouse models or specific compounds that selectively eliminate senescent cells in vivo (known as senolytic strategies) have underscored the detrimental role of senescence in physiological aging and age-related pathologies.172,173 These studies have moved away from the original paradigm that considered senescence as a mainly beneficial cellular mechanism aimed at constraining the damage that cannot be repaired through stable cell cycle arrest. Senescence is induced by telomere attrition, DNA damage, or other stress signals that trigger the activation of antiproliferative pathways, such as p16Ink4a-Rb and p19Arf-p53-p21, leading to growth arrest.174 On senescence induction, cells secrete numerous proinflammatory cytokines, growth factors, and matrix remodeling enzymes that constitute the so called senescence-associated secretory phenotype.175 In a conciliatory model, senescent cells might contribute to clear damage cells within a tissue through the recruitment of inflammatory agents, favoring wound healing processes, as well as to suppress tumor arising by impairing the propagation of aberrant cells. However, with age, and as a consequence of inefficient clearing or defective regenerative capacity of the tissue, senescent cells accumulate and lead to a chronic inflammatory state that impairs proper tissue function. Therefore, their sustained presence over time may be detrimental and boost the aging process.

The generation of different genetically modified mouse models has contributed enormously to the current understanding of senescence in aging. Thus, engineering mice carrying senescent reporters, mainly based on the expression of p16Ink4a (knockin p16+/LUC and transgenic p16-3MR and p16-INK-ATTAC mice), has allowed the tracking of senescent cells in vivo demonstrating their accumulation over time.176–178 This tracking system was first combined with the expression of an inducible killing cassette driven by p16Ink4a to selectively eliminate senescent cells in Bub1bH/H progeroid mice, providing evidence that the accumulation of senescent cells was deleterious in a context of accelerated aging.25 Previously, conventional strategies based on the generation of double deficient mice that lacked either p16Ink4a or p53 in combination with the targeted gene had also demonstrated an improvement in healthspan and lifespan of different progeroid mice models. Thus, Bmi1−/− mice, whose phenotype is mainly driven by the impairment of the self-renewal capacities of different stem cell compartments, showed a significant recovery on mating with mice deficient in the Ink4a/Arf locus.100,179,180 However, Zmpste24−/− mice lived longer when removing p53.60 Likewise, both XpdTTD/TTD progeroid and naturally aged mice show improved healthspan when treated with a peptide that excludes p53 from its nuclear localization hampering its activity.181 However, in other progeroid mouse models, such as those deficient in Ku80 (Xrcc5−/−) or Brca1 (Brca1Δ11/Δ11), the elimination of p53 rescued senescent and premature aging features but significantly increased their tumor susceptibility.182,183 Furthermore, p53 deletion shortens lifespan of some mice showing accelerated aging phenotypes, indicating that the type and extent of the damage may be critical in determining whether p53-driven senescence or apoptosis may be overall beneficial or detrimental.34,184,185 In addition, mice bearing extra dosage of p19Arf/p53 or p16Ink4a/p19Arf show extended longevity, independent of a higher cancer protection.186,187

Nevertheless and despite the many unanswered questions in this field, recent studies have demonstrated that, in a wild-type context, old mice live longer when p16Ink4a-expressing cells are removed.178 More important, this lifespan extension was not linked to an increase in tumor susceptibility but to a reduction. In addition to healthspan improvement in old mice, genetic or pharmacological senolytic strategies have also demonstrated that the specific elimination of senescent cells prevented age-related osteoarthritis and atherosclerosis and rejuvenated both HSC and muscle stem cell compartments.188–190 However, given that in some processes, such as wound healing and liver fibrosis, the elimination of senescent cells is detrimental, the use of senolysis as an antiaging strategy still has to face many challenges to become a reliable therapy.172,173,177,191

Stem Cell Exhaustion

The decline in stem cell functions with age is well documented.192 The use of aged wild-type mice has allowed a compelling characterization of the molecular and functional changes that adult stem cells undergo over time, exposing their diminished capacity to maintain tissue homeostasis and regenerative potential on injury. Side by side, the use of genetically modified mouse models has uncovered many factors that transform stem cell behavior with age. Moreover, these models have also demonstrated that stem cell rejuvenation can slow down the aging process.193 Conversely, engineered mice whose primary defect can be attributed to a severe stem cell dysfunction in different compartments age prematurely.98,194,195

Mouse models have taught us that perturbations in virtually any of the above-mentioned hallmarks of aging may have a detrimental impact in stem cell properties and ultimately contribute to organismal decline.35,193,196,197 Several of the discussed progeroid mouse models with severe DNA damage accumulation, such as Ercc1−/−, Zmpste24−/−, or Bub1bH/H, show defects in different stem cell compartments.198–203 Also, DNA damage that accumulates in normal aging triggers the proteolysis of type XVII collagen, impairing the maintenance of hair follicle stem cells.204 Moreover, mouse models have demonstrated that stem cells are exquisitely sensitive to telomere attrition. Thus, manipulation of telomere length has clear consequences on the functionality of stem cells and the speed of the aging process.67,75,78 Mice deficient in histone remodeling enzymes, such as Bmi1−/− or Sirt6−/−, also exhibit stem cell defects caused by impaired epigenetic regulation of different signaling pathways.93,99,100 In addition, the premature aging defects observed in Arhgap1−/− HSCs have been attributed to alterations in their epigenetic marks.32 Similarly, the maintenance of proteostasis through effective clearance of damaged proteins and organelles has been shown to be essential to preserve the function of old HSCs and muscle stem cells, thanks to the generation of mice deficient in key autophagy genes.118,119

Besides the primary hallmarks, alterations in the so called antagonistic hallmarks—which may have opposite effects on the aging process depending on their intensity3—also affect stem cell behavior. In agreement with the general paradigm (see Deregulated Nutrient Sensing), rapamycin treatment rejuvenates HSCs.205 However, the generation of genetically modified mouse models targeting the mTOR pathway demonstrated that complete loss of mTOR activity can be also detrimental because HSCs require a fine regulation of protein synthesis for a proper maintenance.206,207 Mitochondrial metabolism also influences stem cell aging. As a consequence, simultaneous deletion of 3 FOXO transcription factors, which regulate the resistance to oxidative stress, led to defects in the regenerative potential of HSCs that could be reverted with antioxidant treatment.208 Likewise, improvement of mitochondrial function rejuvenates muscle stem cells in aged mice.159 Finally, as discussed in the previous section, a variety of genetically modified mouse models have demonstrated that cellular senescence impairs stem cell function with age.209–212

Despite the evidenced role of cell intrinsic factors in the modulation of stem cell function, stem cell behavior is intimately influenced by changes in its niche and by the action of secreted factors that can act systemically. The role of these cell-extrinsic regulators and their effect on the aging process will be discussed in the next section.

Altered Intercellular Communication

If we think of aging as a global organismal decline, it is impossible to dismiss the role that cell-extrinsic environment plays in this complex process given that we are interconnected multicellular organisms. Once cells age, the amount and type of secreted factors (including hormones, metabolites, and inflammatory agents) undergo extensive changes and influence the function of neighbor and distant cells, which in turn may respond altering their activity and secretome, thereby generating a deleterious vicious cycle. The best example of the influence of cell-extrinsic factors on the aging process can be found in the effect that the external milieu exerts on the stem cells. Thus, a pioneer work from Conboy et al213 with mouse models of heterochronic parabiosis (where 2 mice of different age share their circulatory systems) showed that the exposure to a young blood was enough to rejuvenate the muscle stem cell function of the old pair. Conversely, young stem cells underwent a functional decline after exposure to an old environment, indicating that proaging factors are also produced over time.213,214 More important, these experiments have demonstrated that, besides the effect on the specific stem cell compartment, it is also possible to reverse the decline of the tissue function.215 Moreover, it has been shown that transplantation of muscle stem cells to a progeroid mouse model exerted a therapeutic effect on distant sites from the area of transplant, indicating that this improvement was mediated by secreted factors. Of note, this approach was able to extend the lifespan of the progeroid mice.199 Likewise, mosaic mice where progeroid Zmpste24-deficient cells coexist with Zmpste24-proficient cells in similar proportion show a complete reversion of the phenotype, supporting the involvement of structural and diffusible factors in the phenotypic rescue.216

Accordingly, there have been described several mouse models with extended lifespan whose increased longevity has been attributed to circulating factors. Thus, mice overexpressing klotho, a pleiotropic circulating protein that is cleaved from its transmembrane form, live significantly longer than their wild-type littermates and show enhanced cognition.217,218 Conversely, mice deficient in klotho display accelerated aging phenotypes and reduced lifespan.219,220 Klotho has been shown to suppress IIS pathway and to regulate Wnt signaling, among other functions.217–221 Similarly, overexpression of the hepatic hormone FGF21 (fibroblast growth factor 21) extends lifespan in mice by blunting the GH/IIS pathway in the liver.222 Indeed, the role of this pathway in aging (extensively discussed in Deregulated Nutrient Sensing) can be also analyzed through the prism of intercellular communication.

The brain, as a master organizer of the functioning of an organism, has also been demonstrated to play a key role in the aging process acting on peripheral tissues. As mentioned above, BRASTO mice (which overexpress Sirt1 in the brain) show a significant improvement in their muscle function and a delay in several age-related phenotypes, together with extended longevity.88 Likewise, overexpression of the uncoupling protein 2 in the hypothalamus triggered an elevation of the temperature in the local area that was counteracted by a reduction in the core body temperature of the mice. This global effect on the entire organism led to an increase in energy efficiency and lifespan.223 Conversely, decreasing the expression of the pain receptor Trpv1 (transient receptor potential vanilloid 1) in the brain reduced the production of the neuropeptide Cgrp, which in turn favored insulin secretion in the pancreas, a youthful metabolic profile and a lifespan extension in the mice.224 Altogether, these studies establish a complex crosstalk between the central nervous system and the periphery that encompasses a wide variety of signals with a global effect on metabolic health and longevity. Another example of integrative regulation at an organismal level can be found in the circadian clock. Interestingly, changes in circadian rhythmicity have been detected as we age.225 In addition, mice deficient in components of the circadian loop, such as Bmal1−/− mice, show accelerated aging and reduced lifespan.226,227 However, many of these phenotypes were not observed when Bmal1 was deleted in the adulthood despite a confirmed disruption of core clock function.228 Moreover, there is a large variation among the aging phenotypes (if any) of the mutant mice that have been generated for different components of the circadian clock, suggesting that further investigation is needed to determine whether the observed phenotypes are truly a consequence of perturbations in circadian rhythmicity or the result of expression changes in specific genes that are under their regulation.229

Inflammation is another relevant example of altered intercellular communication that influences the aging process. Thus, aging is characterized by the existence of a chronic low-grade inflammation, named inflammaging and triggered by diverse stimuli, such as the accumulation of senescent cells or the existence of a dysfunctional immune system.230,231 Accordingly, different mouse models of accelerated aging show exacerbated inflammatory condition through enhanced NF-κB signaling, whose inhibition significantly improved the progeroid phenotype and even extended mice lifespan, supporting the contribution of chronic inflammation to the aging process.232,233 In agreement, mice deficient in the p50 subunit of the NF-κB transcription factor (Nfkb1−/−) exhibit progressive low-grade inflammation and a premature aging phenotype, which leads to decreased longevity.234,235 Conversely, inhibition of NF-κB signaling through genetic manipulation in the hypothalamus extended longevity in mice.236 Moreover, this work demonstrated that NF-κB activation reduced gonadotropin-releasing hormone production, and the decline in this hormone contributed to the development of age-related phenotypes, further supporting the interconnection among a variety of signaling circuits with a global effect at the organismal level. In addition, mice deficient in the anti-inflammatory cytokine interleukin 10 show a wide range of age-related features, including loss of muscle strength, osteopenia, and impaired cardiac function, being considered a mouse model for human frailty.237–240

Conclusions and Remarks

In this review, we have evaluated the 9 hallmarks of aging through the lens of the information provided by the large number of genetically modified mouse models that have been generated during the past decades. This information is invaluable because it provides a causal evidence of the contribution of a specific hallmark to the aging process, beyond any correlative analyses. Indeed, by definition, an ideal hallmark should fulfill the criteria that its experimental aggravation should accelerate aging whereas its amelioration should retard the organismal decline.3 Therefore, gain- and loss-of-function mouse models stand out as a good tool to appraise these criteria. On this basis, we have found examples that meet these guidelines in the majority of hallmarks (Tables 1 and 2). However, this analysis has also surfaced some weaknesses and many challenges ahead.

Table 1. Genetically Engineered Mouse Models With Accelerated Aging Phenotypes and Shortened Lifespan Mouse Model Alternative Nomenclature Gene Targeting Human Syndrome Hallmark References Ercc1−/− Ercc1 knockout XFE progeroid syndrome Genomic instability 11,19 Ercc1−/Δ7 Ercc1 hypomorphic Genomic instability 14 Ercc2R722W/R722W XpdTTD/TTD Ercc2 knockin Trichothiodystrophy Genomic instability 12 Ercc4m/m Xpfm/m Ercc4 knockout Xeroderma pigmentosum group F Genomic instability 13 Ercc5−/− Xpg−/− Ercc5 knockout Xeroderma pigmentosum group G/Cockayne syndrome Genomic instability 16 Ercc6m/m/Xpa−/− Csbm/m/Xpa−/− Double Ercc6/Xpa knockout Cockayne syndrome Genomic instability 7 Xrcc5−/− Ku80−/− or Ku86−/− Xrcc5 knockout Genomic instability 21 Xrcc6−/− Ku70−/− Xrcc6 knockout Genomic instability 21 Prkdc−/− Xrcc7−/−or DNA-PKcs−/− Prkdc knockout Genomic instability 22 Wrn−/−/Terc−/− Double Wrn/Terc knockout Werner syndrome Genomic instability 23 Bub1bH/H BubR1H/H Bub1b hypomorphic Genomic instability 24 Bub1b+/GTTA BubR1+/GTTA Bub1b knockin Mosaic variegated aneuploidy syndrome Genomic instability 27 Bub3+/−/Rae1+/− Double Bub3/Rae1 haploinsufficient Genomic instability 29 SprtnH/H Sprtn hypomorphic Ruijs-Aalfs syndrome Genomic instability 30 Arhgap1−/− Cdc42GAP−/− Arhgap1 knockout Genomic instability 31 AtrS/S Atr hypomorphic Seckel syndrome Genomic instability 34 Atrflox/−:Cre-ERT2+ Atr inducible knockout Genomic instability 35 PolgD257A/D257A mtDNA mutator mouse Polg knockin Genomic instability 38,39 LmnaG609G/G609G LAKI mouse Lmna knockin Hutchinson-Gilford progeria syndrome Genomic instability 55 LmnaL530P/L530P Lmna knockin Hutchinson-Gilford progeria syndrome Genomic instability 53 LmnaHG/+ Lmna knockin Hutchinson-Gilford progeria syndrome Genomic instability 54 Zmpste24−/− Zmpste24 knockout Hutchinson-Gilford progeria syndrome Genomic instability 52 Terc−/− Terc knockout Dyskeratosis congenita Telomere attrition 67 TertER Tert knockin Dyskeratosis congenita Telomere attrition 76 Tert−/− Tert knockout Dyskeratosis congenita Telomere attrition 77 Sirt6−/− Sirt6 knockout Epigenetic alterations 89 Bmi1−/− Bmi1 knockout Epigenetic alterations 98 Stub1−/− CHIP−/− Stub knockout Loss of proteostasis 113 Tg-β5t Ubiquitous β5t overexpression Loss of proteostasis 114 Psme3−/− REGγ−/− Psme3 knockout Loss of proteostasis 115 Gsk3a−/− Gsk3a knockout Loss of proteostasis 241 Sqstm1−/− p62−/− Sqstm1 knockout Mitochondrial dysfunction 161 Cisd2−/− Cisd2 knockout Wolfram syndrome 2 Mitochondrial dysfunction 162 Htra2mnd2/mnd2/Tg-Htra2 Htra2 knockout with rescued Htra2 overexpression in the brain Mitochondrial dysfunction 242 Trp63−/− TAp63−/− Trp63 knockout Stem cell exhaustion 194 Cdk7lox/lox:Ub-CreERT2+/T Cdk7 inducible knockout Stem cell exhaustion 195 Klkl/kl Klothokl/kl Kl knockout Altered intercellular communication 219 Bmal1−/− Bmal1 knockout Altered intercellular communication 226 Nfkb1−/− Nfkb1 knockout Altered intercellular communication 234,235 Il10tm/tm Frail mouse Il10 knockout Altered intercellular communication 239

Table 2. Genetically Engineered Mouse Models With Extended Lifespan Mouse Model Alternative Nomenclature Gene Targeting Hallmark References Tg-Bub1b Ubiquitous Bub1b overexpression Genomic instability 28 Tg-p53/Tg-p16/Tg-p19/Tg-Tert Superp53/superp16/superArf/TgTert Ubiquitous Tert, p53, p16 and p19 overexpression Telomere attrition 79 Tg-Sirt6 Ubiquitous Sirt6 overexpression Epigenetic alterations 92 HttΔQ/ΔQ Htt knockin Loss of proteostasis 116 Tg-Atg5 Ubiquitous Atg5 overexpression Loss of proteostasis 117 Prop1df/df Ames dwarf mouse Prop1 knockin Deregulated nutrient sensing 127 Pou1f1dw/dw Snell dwarf mouse Pouf1f1 knockin Deregulated nutrient sensing 128 Ghrh−/− Ghrh knockout Deregulated nutrient sensing 131 Ghrhrli/li Little mouse Ghrhr knockin Deregulated nutrient sensing 128 Ghr−/− Laron mouse Ghr knockout Deregulated nutrient sensing 130 Pappa−/− Pappa knockout Deregulated nutrient sensing 133 Insrflox/flox:aP2-Cre FIRKO mouse Fat-specific Insr knockout Deregulated nutrient sensing 134 Igfr1flox/flox:Nes-Cre Brain-specific Igfr1 knockout Deregulated nutrient sensing 243 Irs1−/− Irs1 knockout Deregulated nutrient sensing 135 Irs2+/− Irs2 haploinsufficient Deregulated nutrient sensing 136,141* Irs2flox/flox:Nes-Cre Brain-specific Irs2 knockout Deregulated nutrient sensing 136 Igf1m/m Igf1 hypomorphic Deregulated nutrient sensing 137* Igf1r+/− Igf1r haploinsufficient Deregulated nutrient sensing 138,139* Pik3caD933A/+ Pik3ca hypomorphic Deregulated nutrient sensing 143 Tg-Pten Ubiquitous Pten overexpression Deregulated nutrient sensing 144 Akt1+/− Akt1 haploinsufficient Deregulated nutrient sensing 145 MtorΔ/Δ Mtor hypomorphic Deregulated nutrient sensing 150 Mtor+/−/Mlst8+/− Double Mtor/Mlst8 haploinsufficient Deregulated nutrient sensing 149 Rps6kb1−/− Rps6kb1 knockout Deregulated nutrient sensing 148 Myc+/− Myc haploinsufficient Deregulated nutrient sensing 151 mTg-Cat mCAT Mitochondrial-specific Cat overexpression Mitochondrial dysfunction 164 Gpx4+/− Gpx4 haploinsufficient Mitochondrial dysfunction 166 Clk1+/− Clk1 haploinsufficient Mitochondrial dysfunction 167 Surf1−/− Surf1 knockout Mitochondrial dysfunction 168 Tg-p19/Tg-p53 SuperArf/superp53 Ubiquitous p19 and p53 overexpression Cellular senescence 186 TgTg-p16/p19 Ubiquitous p16 and p19 overexpression Cellular senescence 187 Tg-p16-INK-ATTAC p16-driven senolytic cassette overexpression Cellular senescence 178 Tg-Kl Ubiquitous Kl overexpression Altered intercellular communication 217 Tg-Fgf21 Ubiquitous Fgf21 overexpression Altered intercellular communication 222 bTg-Sirt1 BRASTO mouse Brain-specific Sirt1 overexpression Altered intercellular communication 88 bTg-Ucp2 Brain-specific Ucp2 overexpression Altered intercellular communication 223 bTg-Trpv1 Brain-specific Trpv1 overexpression Altered intercellular communication 224 Ikbkblox/lox:Nes-Cre Brain-specific Ikbkb knock-out Altered intercellular communication 236 bTg-Plau α-MUPA mouse Brain-specific Plau overexpression Altered intercellular communication 244 p66−/− p66 knockout 245 Mtbp+/− Mtbp haploinsufficient 246

Among primary hallmarks, which are those unequivocally negative, there is a broad number of mouse models with a wide range of genomic defects that show accelerated aging, emphasizing the idea that DNA damage accumulation is detrimental for the aging process. However, despite confirmed evidence that genetic lesions accumulate with age, therefore meeting the third and last criterion to be an aging hallmark, the amount and type of damage found in these mouse models may differ from what occurs during healthy aging. Of note, Polg+/D257A mice exhibit an mtDNA mutation frequency 30-fold higher than an old mice and show no overt phenotype. Nevertheless, the finding that overexpression of a protein responsible for proper chromosomal segregation (BubR1) extends lifespan suggests that genomic instability can be indeed a real life limiting factor. Accordingly, more research is needed to evaluate the type and threshold of DNA damage that contributes to normal aging and to explore whether boosting the DNA repair machinery becomes an effective antiaging strategy. In agreement, reintroducing telomerase activity at an old age has been shown to extend longevity without increasing tumor susceptibility. Following on the primary hallmarks, additional weaknesses have been found when analyzing the mouse models that show epigenetic alterations. Thus, there is a great difference between the amount of gain- and loss-of-function models that modulate longevity in invertebrates and those that do so in mice. Besides, the information generated by these mouse models suggests that opposite changes in global epigenetic patterns may lead to similar deleterious outcomes, indicating that more effort should be made toward identifying the specific loci that underlie the aging phenotype in each case. Similarly, although there are many mouse models supporting the role of proteostasis in age-related diseases, few have demonstrated that a failure in the machinery aimed at maintaining protein homeostasis accelerates healthy aging. Indeed, some of these few examples have been linked to a defect in the processing of a specific protein rather than to a global accumulation of damaged/misfolded proteins. An explanation for the limited data generated to date on longevity is that the deficiency of some relevant genes in these pathways has turned out to be pathological (or even lethal) in several cases or, by contrast, has unveiled certain functional redundancy among components of the proteostasis network. Nevertheless, the lifespan extension reported on Atg5 overexpression paves the way to explore the potential for proteostasis improvement as a strategy to slow down the aging process.

With regards to antagonistic hallmarks, mouse models of mitochondrial dysfunction and cellular senescence fit well with the definition. Thus, genetically engineered mice have demonstrated that both senescence and even certain level of mitochondrial dysfunction can be beneficial for the organism, but, over a threshold, the effect on the aging process becomes detrimental. Interestingly, nutrient sensing has also been categorized as an antagonistic hallmark, although the vast majority of mouse models support the view that by blunting IIS and anabolism is possible to increase longevity. Nevertheless, these pathways are necessary for survival, and therefore they are considered as antagonistic. In this sense, it is important to underscore that an excessive IIS suppression is observed in certain progeroid models, and IGF-1 supplementation ameliorates the aging phenotype. Accordingly, it would be interesting to know whether the prolongevity effect remains if the genetic manipulation on these pathways is performed in old mice rather than at a young stage. This information would help to better define the effect of metabolic interventions in the elderly.

Finally, within the last category of integrative hallmarks, which groups stem cell exhaustion and intercellular communication, we have found good examples evidencing that a deterioration in stem cell function or a global low-chronic inflammatory condition lead to an organismal decline. Likewise, mouse models have demonstrated that this decline is indeed the result of the accumulation of different types of damage that, in an integrative manner, negatively impact on the entire organism. Nevertheless, mouse models have also provided proof-of-principle that interventions aimed at rejuvenating stem cells or improving cellular and tissue interconnections are among the most effective strategies to modulate healthspan and longevity.

Nonstandard Abbreviations and Acronyms GH growth hormone HSC hematopoietic stem cell IGF-1 insulin-like growth factor 1 IIS insulin and insulin-like growth factor 1 signaling pathway mtDNA mitochondrial DNA mTOR target of rapamycin NER nucleotide excision repair ROS reactive oxygen species

Sources of Funding The Instituto Universitario de Oncología del Principado de Asturias is supported by Fundación Bancaria Caja de Ahorros de Asturias. Our work is supported by grants from European Research Council (DeAge, ERC Advanced Grant), Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (RTICC), and Progeria Research Foundation. A.R. Folgueras is recipient of a Ramón y Cajal Fellowship. S. Freitas-Rodríguez is recipient of an FPU (Formación de Profesorado Universitario) Fellowship.

Disclosures None.

Footnotes