Studying long-lived animals provides novel insight into shared characteristics of aging and represents a unique model to elucidate approaches to prevent chronic disease. Oxidant stress underlies many chronic diseases and resistance to stress is a potential mechanism governing slowed aging. The transcription factor nuclear factor (erythroid-derived 2)-like 2 is the “master regulator” of cellular antioxidant defenses. Nrf2 is upregulated by some longevity promoting interventions and may play a role in regulating species longevity. However, Nrf2 expression and activity in long-lived models have not been well described. Here, we review evidence for altered Nrf2 signaling in a variety of slowed aging models that accomplish lifespan extension via pharmacological, nutritional, evolutionary, genetic, and presumably epigenetic means.

1. Introduction

The incidence of chronic disease increases with age. Understanding the relationships between the processes of aging and age-related diseases is an important initiative of the National Institutes of Health to improve the health of the aging population [1]. Slowing the aging process limits the burden of age-related chronic disease [2]. Identifying characteristics that slow aging may also provide approaches for preventing chronic diseases. Animals with increased lifespan aid in understanding the aging process by allowing the study of physiological and biochemical adaptations associated with slowed aging. Further, studying characteristics shared among long-lived models provides insight into pathways that are key to slowing the aging process and age-associated chronic diseases.

Lifespan can be extended by genetic, dietary, and pharmacological interventions. Additionally, multiple species have independently evolved long lifespan, including humans and naked mole rats, both of which live more than four times longer than predicted by body size [3]. Some of the earliest discoveries of lifespan extension were single-gene mutations associated with the insulin-like growth factor I (IGF-1) and growth hormone (GH) pathways. These mice, including the Snell dwarf [4], are smaller than their heterozygote counterparts and significantly longer-lived, some by 40% or more compared with controls. Long-term caloric restriction is the most consistent dietary manipulation to extend lifespan and recent evidence suggests that short-term transient nutrition restriction prior to weaning, accomplished by litter enlargement, also increases mean and maximal lifespan in mice [5]. Pharmaceutical manipulation of lifespan is in its infancy, with evidence that rapamycin can extend lifespan in mice [6].

It is well established that oxidant stress increases with age across a variety of tissues, including cardiac [7] and skeletal muscle [8], liver [9], and brain [10], and is associated with a wide variety of chronic age-related diseases including cancer, neurodegeneration, sarcopenia, and cardiovascular disease. Although the oxidative stress theory of aging has received criticism [11], it remains true that oxidant stress is associated with the aging process. In response to oxidative stress, cells upregulate antioxidant pathways, including activation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2), the master regulator of antioxidant defenses and the proposed “master regulator” of the aging process [3]. Further, the therapeutic potential of Nrf2 is well supported in neurodegeneration and cancer (reviewed in [12, 13]), highlighting a role for Nrf2 in attenuating age-related chronic disease. Below, we will review what is known about Nrf2 in four models of lifespan extension: caloric restriction, rapamycin feeding, short-term nutrition restriction, and the Snell dwarf mouse. Further, we will discuss what is known about Nrf2 in the exceptionally long-lived naked mole rat and in humans who show enhanced longevity, with the overall goal of describing Nrf2 signaling in longevity interventions and in naturally occurring models of long life.

2. Nrf2 Signaling Basics

A member of the basic leucine zipper transcription factor family, Nrf2, controls both basal and inducible expression of over 200 target genes. When cellular stress is low, Nrf2 is sequestered in the cytoplasm by its involvement in an inactive complex with the actin-binding protein Kelch-like ECH-associated protein 1 (Keap1). Under these conditions, Keap1 targets Nrf2 for ubiquitination and degradation by the 26S proteasome system, resulting in basal low-level expression of Nrf2 [14]. However, when activated, Nrf2 translocates to the nucleus and transcriptionally upregulates its cytoprotective transcriptional program through binding to the antioxidant response element (ARE) in the promoter region of its target genes. Activation by reactive oxygen species (ROS) is the best understood mechanism of Nrf2 activation. Oxidant exposure modifies cysteine residues on Keap1 resulting in conformational changes that protect Nrf2 from targeting for ubiquitination and degradation [15], thus resulting in Nrf2 accumulation and activation. In addition to ROS and electrophilic species, Nrf2 can also be activated by phytochemicals [16–18], as well as various pharmaceuticals (reviewed in [19]) via overlapping and distinct mechanisms.

Nrf2 target genes exhibit antioxidant properties and facilitate cellular responses against xenobiotics. Antioxidant enzymes include NAD(P)H dehydrogenase quinone 1 (NQO1), heme oxygenase-1 (HO-1), peroxiredoxin 1 (Prdx1), superoxide dismutase-1 (SOD-1), and many enzymes involved in glutathione synthesis such as glutathione S-transferases (GSTs) and glutamate-cysteine ligase modifier (GCLM), the rate-limiting step in glutathione synthesis (reviewed in [20]). In addition, the cytoprotective properties of Nrf2 activation extend beyond these classic target genes, as other ARE-containing genes exhibit anti-inflammatory activity [21] and autophagic properties [22] and aid in proteasomal removal of oxidative damaged proteins [23]. Nrf2 also regulates its own expression. Two ARE-like motifs in the 5′flanking region of the Nrf2 promoter are responsible for the induction of Nrf2 upon activation [24], ensuring a feed-forward process with Nrf2 activation promoting its own expression and thus facilitating a profound cellular response to stress.

Studies of Nrf2 knockout animals highlight the importance of Nrf2 in cytoprotection. These animals display a diminished ability to activate prosurvival genes [30] and are more susceptible to a wide range of stressors including hyperoxia, lipopolysaccharide, cigarette smoke, and UV irradiation, as well as various chemical insults [31]. Nrf2 knockout animals display diminished basal activity of Nrf2-regulated antioxidant enzymes [30], as well as inducible targets. In a transcriptional analysis of Nrf2 target genes, treatment with the Nrf2 activator 3H-1,2-dithiole-3-thione (D3T) induced 292 genes in wild type animals, compared to only 15 in Nrf2 knockout mice [32]. Together, investigations of Nrf2 knockout animals demonstrate diminished basal and inducible gene programs, as well as increased sensitivity to cell stresses, and support Nrf2 as an important transcriptional regulator of the cytoprotective program.

3. Nrf2 and Aging

The role of Nrf2 in responding to cytotoxic stressors is well defined. However, only within the last few years have studies elucidated how Nrf2 function changes with age and how changes in Nrf2 activity contribute to the aging phenotype. Aging is sufficient to diminish cardiac Nrf2-ARE binding activity [7], and aged Nrf2 knockout mice exhibit decreased expression of antioxidant target genes [8]. Disruptions in Nrf2-Keap1 signaling have been reported in skeletal muscle from sedentary older humans [33] and cardiac muscle from aging rats [7]. Impaired liver Nrf2 transcriptional activity in old rats results in glutathione depletion and significant downregulation of Nrf2-regulated glutathione biosynthetic enzymes [34]. Aged mice show similar losses in cellular redox capacity to those observed in Nrf2 knockout mice [34, 35], suggesting that Nrf2 dysregulation with age may be responsible for the loss of cellular redox status. Diminished Nrf2 target gene expression with age is accompanied by increased muscle ROS production, glutathione depletion, and increased oxidant damage to proteins, DNA, and lipids in both humans [33] and rodents [34]. Therefore, given that Nrf2 activity decreases with age alongside increased oxidant stress, interventions that activate Nrf2 may impact the aging process and longevity.

Support for the role of Nrf2 in regulation of lifespan comes from Nrf2 gain of function and loss of function studies. For example, experimental deletion of the antielectrophilic gene glutathione transferase (gGsta4) activated Nrf2 and significantly extended lifespan in mice [36]. This mutation increased electrophilic lipid peroxidation products and increased nuclear Nrf2 activity by 43% and 38% in liver and skeletal muscle, respectively. The authors propose that deletion of this glutathione transferase gene resulted in chronic moderate Nrf2 activation and presumably elevated downstream Nrf2 signaling throughout the mouse lifespan. Studies of the Nrf2 homolog SKN-1 in Caenorhabditis elegans (C. elegans) and the Drosophila homolog CncC further suggest that Nrf2 may be implicated in longevity processes. Upon activation, SKN-1 and CncC upregulate genes involved in the oxidative stress response, including many orthologs to those regulated by mammalian Nrf2 [37, 38]. Similar to mouse Nrf2 knockouts, SKN-1 mutants show diminished resistance to oxidative stress and shortened lifespan. On the other hand, moderate overexpression of a constitutively active SKN-1 increases lifespan, alongside increased resistance to oxidative stress [37]. Similarly, Keap1 loss-of-function mutations extend the lifespan of male Drosophila [38]. Various lifespan extending genetic manipulations in C. elegans require SKN-1. Dietary restriction activates SKN-1, and expression of the transcription factor in C. elegans neuronal cells is required for longevity to be extended by dietary restriction [39]. The long-lived daf-2 (nematode homologue of FOXO) mutant increases lifespan in part through resultant activation of SKN-1 [40]. Together, what is known about Nrf2 and aging, alongside preliminary studies of the role of Nrf2 and SKN-1 in cytoprotection, suggests that loss of Nrf2 is important in age-associated declines in oxidant stress resistance, and perhaps in the aging pathology itself.

4. Nrf2 in Long-Lived Models

4.1. Naked Mole Rat

The naked mole rat is an exceptionally long-lived species, with a lifespan four times longer than similarly sized rodents, thus making the naked mole rat an important model for longevity studies [41]. Naked mole rats do not have typical lifespan curves in which mortality rates increase with age, but rather they experience few of the biological changes typically associated with aging such as decreased metabolic rate, body composition changes, and declines in genomic and proteomic integrity [42]. Naked mole rats are resistant to age-associated diseases such as cancer [43], cardiac diastolic dysfunction [44], and neurodegenerative diseases [45]. Thus, the naked mole rat represents a naturally occurring, unique model of healthy aging.

Surprisingly, early studies of naked mole rats revealed that they have higher levels of oxidative damage, including lipid peroxidation products, protein carbonyls, and oxidative DNA modification in the liver, compared to shorter-lived mouse controls [46]. Subsequent comparisons of young and old naked mole rats revealed a striking difference between age-associated changes in oxidative stress markers [47]. While macromolecular oxidant damage increases with age in mice, as it does in humans, naked mole rats maintain high levels of oxidative damage throughout their lifespan, similar to those observed in old mice [47]. In fact, few genes show differential expression between young and old animals, as assessed by transcriptome analyses in the brain, liver, and kidney from 4- and 20-month-old naked mole rats [26]. Therefore, while naked mole rats from a young age contain higher levels of oxidative damage than shorter-lived control mice, the typical age-associated increase in damage is blunted in this species, suggesting maintenance of oxidant stress defenses over time.

Naked mole rats also have significantly elevated proteasome quality control mechanisms [25]. The high breakdown and clearance of damaged proteins is suspected to be largely due to increased Nrf2 expression, as Nrf2 regulates the transcription of α and β subunits of the 26S proteasome, as well as the selective autophagy cargo protein p62 [48–50]. In support of the hypothesized role of Nrf2 in naked mole rat longevity, under nonstressed conditions, naked mole rats have greater protein levels of Nrf2 including nuclear Nrf2, elevated Nrf2-ARE binding activity, and greater expression of Nrf2-regulated enzymes in fibroblasts and liver [51, 52]. These data suggest Nrf2 may be responsible for the heightened quality control mechanisms in naked mole rats and may be associated with their exceptional longevity.

4.2. Caloric Restriction

Caloric restriction (CR), a decrease in caloric intake without malnutrition, is the most consistent and robust means to increase lifespan across species, from flies to rodents [53], as well as nonhuman primate models [54]. Additionally, CR imparts slowed aging effects and delays the incidence of age-related disease [55]. Although the mechanisms underlying the effects of CR on longevity remain largely unknown, protection against carcinogenesis [56], reduced insulin/insulin-like growth factor (IGF) signaling [57], and prolonged survival [53] are documented. In addition, CR improves cellular adaptation to stress [58], as evidenced by liver mitochondria isolated from CR rats which show delayed opening of the mitochondrial transition pore upon oxidative challenge [58]. In an elegant study using sera collected from humans practicing long-term CR compared to sera collected from age- and sex-matched individuals following a typical western diet, treatment of cultured human primary fibroblasts with CR sera significantly upregulated gene expression of stress-response genes and enhanced tolerance to oxidants [59]. CR decreases ROS production, enhances the plasma membrane redox system, improves insulin signaling, and attenuates inflammation [27], all of which have been associated with improved age-related disease outcomes.

Many of the positive outcomes of CR have been associated with Nrf2 activation. For example, a variety of carcinogens activate Nrf2 and the ARE, protecting against carcinogenesis [60]. Changes in insulin levels, such as those elicited by fasting, elicit a small acute oxidant stress and subsequent activation of Nrf2 and its targets [61]. CR prevents the age-induced loss of cellular antioxidant capacity, in part due to increased levels of Nrf2 targets NQO1 and GSTs in brain and liver [62, 63]. In cerebral vascular endothelial cells, CR prevents the age-related decline in Nrf2 activity. Further, CR upregulates Nrf2 expression in old mice to a level that surpasses that of young ad libitum (AL) fed animals [64], highlighting the importance of Nrf2 with aging and its potential activation by longevity promoting interventions. Several proposed CR mimetics, such as resveratrol, quercetin, and curcumin [65], act to increase lifespan and slow aging at least in part through activation of Nrf2 [28, 66–68]. We assessed Nrf2 signaling in cardiac muscle from 7-month-old animals that underwent lifelong 40% reduction in caloric intake compared to mice fed AL. We found Nrf2 protein expression to be unchanged by CR. However, NQO1 and SOD-1 were significantly upregulated (Figure 1) in CR, suggesting that a lifelong reduction in caloric intake may activate Nrf2 and be implicated in longevity in these animals.