Kaitlyn N. Lewis

Nrf2, a Guardian of Healthspan and Gatekeeper of Species Longevity

Although aging is a ubiquitous process that prevails in all organisms, the mechanisms governing both the rate of decline in functionality and the age of onset remain elusive. A profound constitutively upregulated cytoprotective response is commonly observed in naturally long-lived species and experimental models of extensions to lifespan (e.g., genetically-altered and/or experimentally manipulated organisms), as indicated by enhanced resistance to stress and upregulated downstream components of the cytoprotective nuclear factor erythroid 2-related factor 2 (Nrf2)-signaling pathway. The transcription factor Nrf2 is constitutively expressed in all tissues, although levels may vary among organs, with the key detoxification organs (kidney and liver) exhibiting highest levels. Nrf2 may be further induced by cellular stressors including endogenous reactive-oxygen species or exogenous electrophiles. The Nrf2-signaling pathway mediates multiple avenues of cytoprotection by activating the transcription of more than 200 genes that are crucial in the metabolism of drugs and toxins, protection against oxidative stress and inflammation, as well as playing an integral role in stability of proteins and in the removal of damaged proteins via proteasomal degradation or autophagy. Nrf2 interacts with other important cell regulators such as tumor suppressor protein 53 (p53) and nuclear factor-kappa beta (NF-κB) and through their combined interactions is the guardian of healthspan, protecting against many age-related diseases including cancer and neurodegeneration. We hypothesize that this signaling pathway plays a critical role in the determination of species longevity and that this pathway may indeed be the master regulator of the aging process.

Regulation of Nrf2 signaling and longevity in naturally long-lived rodents.

Significance Both genetically altered and naturally long-lived mammals are more resistant to toxic compounds that may cause cancer and age-associated diseases than their shorter-lived counterparts. The mechanisms by which this stress resistance occurs remain elusive. We found that longer-lived rodent species had markedly higher levels of signaling activity of the multifunctional regulator nuclear factor erythroid 2-related factor (Nrf2) and that this increase in cytoprotective signaling appeared to be due to species differences in Kelch-like ECH-Associated Protein 1 (Keap1) and β-transducin repeat-containing protein (βTrCP) regulation of Nrf2 activity. Both of these negative regulators of Nrf2-signaling activity are significantly lower in longer-lived species. By targeting the proteins that regulate Nrf2 rather than Nrf2 itself, we may be able to identify new therapies that impact aging and age-associated diseases such as cancer. The preternaturally long-lived naked mole-rat, like other long-lived species and experimental models of extended longevity, is resistant to both endogenous (e.g., reactive oxygen species) and environmental stressors and also resists age-related diseases such as cancer, cardiovascular disease, and neurodegeneration. The mechanisms behind the universal resilience of longer-lived organisms to stress, however, remain elusive. We hypothesize that this resilience is linked to the activity of a highly conserved transcription factor, nuclear factor erythroid 2-related factor (Nrf2). Nrf2 regulates the transcription of several hundred cytoprotective molecules, including antioxidants, detoxicants, and molecular chaperones (heat shock proteins). Nrf2 itself is tightly regulated by mechanisms that either promote its activity or increase its degradation. We used a comparative approach and examined Nrf2-signaling activity in naked mole-rats and nine other rodent species with varying maximum lifespan potential (MLSP). We found that constitutive Nrf2-signaling activity was positively correlated (P = 0.0285) with MLSP and that this activity was also manifested in high levels of downstream gene expression and activity. Surprisingly, we found that species longevity was not linked to the protein levels of Nrf2 itself, but rather showed a significant (P < 0.01) negative relationship with the regulators Kelch-like ECH-Associated Protein 1 (Keap1) and β-transducin repeat-containing protein (βTrCP), which target Nrf2 for degradation. These findings highlight the use of a comparative biology approach for the identification of evolved mechanisms that contribute to health span, aging, and longevity.

The Naked Mole-Rat Response to Oxidative Stress: Just Deal with It

SIGNIFICANCE The oxidative stress theory of aging has been the most widely accepted theory of aging providing insights into why we age and die for over 50 years, despite mounting evidence from a multitude of species indicating that there is no direct relationship between reactive oxygen species (ROS) and longevity. Here we explore how different species, including the longest lived rodent, the naked mole-rat, have defied the most predominant aging theory. RECENT ADVANCES In the case of extremely long-lived naked mole-rat, levels of ROS production are found to be similar to mice, antioxidant defenses unexceptional, and even under constitutive conditions, naked mole-rats combine a pro-oxidant intracellular milieu with high, steady state levels of oxidative damage. Clearly, naked mole-rats can tolerate this level of oxidative stress and must have mechanisms in place to prevent its translation into potentially lethal diseases. CRITICAL ISSUES In addition to the naked mole-rat, other species from across the phylogenetic spectrum and even certain mouse strains do not support this theory. Moreover, overexpressing or knocking down antioxidant levels alters levels of oxidative damage and even cancer incidence, but does not modulate lifespan. FUTURE DIRECTIONS Perhaps, it is not oxidative stress that modulates healthspan and longevity, but other cytoprotective mechanisms that allow animals to deal with high levels of oxidative damage and stress, and nevertheless live long, relatively healthy lifespans. Studying these mechanisms in uniquely long-lived species, like the naked mole-rat, may help us tease out the key contributors to aging and longevity.

Stress Resistance in the Naked Mole-Rat: The Bare Essentials – A Mini-Review

Background: Studies comparing similar-sized species with disparate longevity may elucidate novel mechanisms that abrogate aging and prolong good health. We focus on the longest living rodent, the naked mole-rat. This mouse-sized mammal lives ∼8 times longer than do mice and, despite high levels of oxidative damage evident at a young age, it is not only very resistant to spontaneous neoplasia but also shows minimal decline in age-associated physiological traits. Objectives: We assess the current status of stress resistance and longevity, focusing in particular on the molecular and cellular responses to cytotoxins and other stressors between the short-lived laboratory mouse and the naked mole-rat. Results: Like other experimental animal models of lifespan extension, naked mole-rat fibroblasts are extremely tolerant of a broad spectrum of cytotoxins including heat, heavy metals, DNA-damaging agents and xenobiotics, showing LD50 values between 2- and 20-fold greater than those of fibroblasts of shorter-lived mice. Our new data reveal that naked mole-rat fibroblasts stop proliferating even at low doses of toxin whereas those mouse fibroblasts that survive treatment rapidly re-enter the cell cycle and may proliferate with DNA damage. Naked mole-rat fibroblasts also show significantly higher constitutive levels of both p53 and Nrf2 protein levels and activity, and this increases even further in response to toxins. Conclusion: Enhanced cell signaling via p53 and Nrf2 protects cells against proliferating with damage, augments clearance of damaged proteins and organelles and facilitates the maintenance of both genomic and protein integrity. These pathways collectively regulate a myriad of mechanisms which may contribute to the attenuated aging profile and sustained healthspan of the naked mole-rat. Understanding how these are regulated may be also integral to sustaining positive human healthspan well into old age and may elucidate novel therapeutics for delaying the onset and progression of physiological declines that characterize the aging process.

Organization of the Mammalian Metabolome according to Organ Function, Lineage Specialization, and Longevity

Biological diversity among mammals is remarkable. Mammalian body weights range seven orders of magnitude and lifespans differ more than 100-fold among species. While genetic, dietary, and pharmacological interventions can be used to modulate these traits in model organisms, it is unknown how they are determined by natural selection. By profiling metabolites in brain, heart, kidney, and liver tissues of 26 mammalian species representing ten taxonomical orders, we report metabolite patterns characteristic of organs, lineages, and species longevity. Our data suggest different rates of metabolite divergence across organs and reveal patterns representing organ-specific functions and lineage-specific physiologies. We identified metabolites that correlated with species lifespan, some of which were previously implicated in longevity control. We also compared the results with metabolite changes in five long-lived mouse models and observed some similar patterns. Overall, this study describes adjustments of the mammalian metabolome according to lifespan, phylogeny, and organ and lineage specialization.

Walking the oxidative stress tightrope: A perspective from the naked mole-rat, the longest-living rodent

Reactive oxygen species (ROS), by-products of aerobic metabolism, cause oxidative damage to cells and tissue and not surprisingly many theories have arisen to link ROS-induced oxidative stress to aging and health. While studies clearly link ROS to a plethora of divergent diseases, their role in aging is still debatable. Genetic knock-down manipulations of antioxidants alter the levels of accrued oxidative damage, however, the resultant effect of increased oxidative stress on lifespan are equivocal. Similarly the impact of elevating antioxidant levels through transgenic manipulations yield inconsistent effects on longevity. Furthermore, comparative data from a wide range of endotherms with disparate longevity remain inconclusive. Many long-living species such as birds, bats and mole-rats exhibit high-levels of oxidative damage, evident already at young ages. Clearly, neither the amount of ROS per se nor the sensitivity in neutralizing ROS are as important as whether or not the accrued oxidative stress leads to oxidative-damage-linked age-associated diseases. In this review we examine the literature on ROS, its relation to disease and the lessons gleaned from a comparative approach based upon species with widely divergent responses. We specifically focus on the longest lived rodent, the naked mole-rat, which maintains good health and provides novel insights into the paradox of maintaining both an extended healthspan and lifespan despite high oxidative stress from a young age.

From yeast to human: exploring the comparative biology of methionine restriction in extending eukaryotic life span

Methionine restriction is a widely reported intervention for increasing life span in several model organisms. Low circulating levels of methionine are evident in the long‐lived naked mole‐rat, suggesting that it naturally presents with a life‐extending phenotype akin to that observed in methionine‐restricted animals. Similarly, long‐lived dwarf mice also appear to have altered methionine metabolism. The mechanisms underlying methionine‐restriction effects on life‐span extension, however, remain unknown, as do their potential connections with caloric restriction, another well‐established intervention for prolonging life span. Paradoxically, methionine is enriched in proteins expressed in mitochondria and may itself serve an important role in the detoxification of reactive oxygen species and may thereby contribute to delayed aging. Collectively, we highlight the evidence that modulation of the methionine metabolic network can extend life span—from yeast to humans—and explore the evidence that sulfur amino acids and the concomitant transsulfuration pathway play a privileged role in this regard. However, systematic studies in single organisms (particularly those that exhibit extreme longevity) are still required to distinguish the fundamental principles concerning the role of methionine and other amino acids in regulating life span.

Unraveling the message: insights into comparative genomics of the naked mole-rat

Animals have evolved to survive, and even thrive, in different environments. Genetic adaptations may have indirectly created phenotypes that also resulted in a longer lifespan. One example of this phenomenon is the preternaturally long-lived naked mole-rat. This strictly subterranean rodent tolerates hypoxia, hypercapnia, and soil-based toxins. Naked mole-rats also exhibit pronounced resistance to cancer and an attenuated decline of many physiological characteristics that often decline as mammals age. Elucidating mechanisms that give rise to their unique phenotypes will lead to better understanding of subterranean ecophysiology and biology of aging. Comparative genomics could be a useful tool in this regard. Since the publication of a naked mole-rat genome assembly in 2011, analyses of genomic and transcriptomic data have enabled a clearer understanding of mole-rat evolutionary history and suggested molecular pathways (e.g., NRF2-signaling activation and DNA damage repair mechanisms) that may explain the extraordinarily longevity and unique health traits of this species. However, careful scrutiny and re-analysis suggest that some identified features result from incorrect or imprecise annotation and assembly of the naked mole-rat genome: in addition, some of these conclusions (e.g., genes involved in cancer resistance and hairlessness) are rejected when the analysis includes additional, more closely related species. We describe how the combination of better study design, improved genomic sequencing techniques, and new bioinformatic and data analytical tools will improve comparative genomics and ultimately bridge the gap between traditional model and nonmodel organisms.

Mechanisms of aging in the naked mole-rat: The case for programmed aging

Maximum lifespan is an important species trait that generally scales with body size. Never the less there are many mammals that deviate from this allometric relationship and live more than double the expected lifespan predicted on the basis of body size. These exceptionally long-lived species appear to defy the expected rate of aging and provide powerful tools with which to elucidate if maximum species lifespan is indeed encoded in the genome; determine possible biological clocks and their downstream molecular mechanisms that may abrogate or regulate rates of aging. One such mammal is the naked mole-rat [NMR]. This, the longest-lived rodent known, lives 8.6-times longer than similar-sized mice, yet it maintains cancer-free, good health for more than 85% of its astonishing 30-year lifespan. NMRs, like other long-lived species, show pronounced resistance to most cellular stressors. This may be due to enhanced cellular protection and/or better maintenance of somatic integrity. In contrast, short-lived species generally direct many of their resources into rapid growth and early reproduction rather than fend off threats to their soma. Although the mechanisms facilitating this species divergence in somatic maintenance are poorly understood, these most likely represent an evolutionary trade-off between partitioning energy and resources into somatic maintenance (thereby contributing to the survival of the individual) versus investments in growth and reproduction and ensuring the rapid attainment of sexual maturity and the long-termsurvival of the species. This species-specific difference in resource and somatic management must be encoded in the genome, thus enabling cells and the organism to mount the appropriate level of cytoprotection, commensurate with their expected longevity. We explore this premise by reevaluating the various theories of aging in the light of what is known from the biology of the longest-living rodent, the naked mole-rat.

A window into extreme longevity; the circulating metabolomic signature of the naked mole-rat, a mammal that shows negligible senescence

Mouse-sized naked mole-rats (Heterocephalus glaber), unlike other mammals, do not conform to Gompertzian laws of age-related mortality; adults show no age-related change in mortality risk. Moreover, we observe negligible hallmarks of aging with well-maintained physiological and molecular functions, commonly altered with age in other species. We questioned whether naked mole-rats, living an order of magnitude longer than laboratory mice, exhibit different plasma metabolite profiles, which could then highlight novel mechanisms or targets involved in disease and longevity. Using a comprehensive, unbiased metabolomics screen, we observe striking inter-species differences in amino acid, peptide, and lipid metabolites. Low circulating levels of specific amino acids, particularly those linked to the methionine pathway, resemble those observed during the fasting period at late torpor in hibernating ground squirrels and those seen in longer-lived methionine-restricted rats. These data also concur with metabolome reports on long-lived mutant mice, including the Ames dwarf mice and calorically restricted mice, as well as fruit flies, and even show similarities to circulating metabolite differences observed in young human adults when compared to older humans. During evolution, some of these beneficial nutrient/stress response pathways may have been positively selected in the naked mole-rat. These observations suggest that interventions that modify the aging metabolomic profile to a more youthful one may enable people to lead healthier and longer lives.