FOXO3, a Resilience Gene: Impact on Lifespan, Healthspan, and Deathspan

Oliver Wendell Holmes is purported to have said, “Old age is always fifteen years older than I am.” Given recent rapid advances in understanding the aging process – from molecular, cellular, tissue, organ, to whole body aging – Holmes appears prescient. There is little doubt that people age at different rates and in different ways (1). If one could indeed remain 15 to 20 years “biologically younger” than their chronological age and delay the onset of aging-related chronic diseases and disability, that would be quite an achievement for the individual and society (2). This, of course, assumes that if lifespan is prolonged, one also compresses morbidity, resulting in a longer healthspan, not a longer “deathspan” (ie, poor quality of life from extra years of severe disease and incapacitation), see Figure 1.

Figure 1.

Healthspan vs. Deathspan. To elaborate on “healthspan” and “deathspan”, the former is the number of years free of major disease and disability, while the latter is years with major chronic disease and/or disability. Deathspan can have major public health implications since these are the years that consume the most healthcare resources (42). Moreover, deathspan cannot be reliably predicted at present. Being able to predict the length of deathspan would offer better potential allocation of resources, while providing a more accurate means of measuring and treating aging-associated diseases. This could help to reduce age-associated morbidities.

Established biomarkers of physiological aging, such as maximal oxygen consumption (max. V0₂) for cardiovascular aging and forced expiratory volume1 (FEV1) for pulmonary aging, have been well studied and decline at a predictable rate in humans (3). Those fortunate enough to have a genetically more resilient physiological system, may start with a higher baseline function and/or undergo functional decline more slowly. Importantly, the biological rate of aging for an individual organ or physiological system, can be decelerated or accelerated by non-genetic factors, such as smoking, poor diet, and lack of exercise, among other modifiable “lifestyle” factors (1). Geroscience is becoming ever closer to having phenotypic tools capable of measuring the rate of human aging at multiple system levels. For example, 258 candidate blood-based biomarkers of aging and age-related disease were reviewed by an expert panel for the Targeting Aging with Metformin (TAME) clinical trial (4). About 10 biomarkers made the cut for consideration based on reliability and feasibility, relevance to aging hallmarks, consistently robust in prediction of outcomes (all-cause mortality, clinical and functional utility), and for potential responsiveness to the study intervention. With accurate and reliable measures of aging processes, gauging the “success” of a biological age-modifying intervention is now within reach.

An individual’s genetic make-up has long been known as a strong predictor of lifespan. For example, offspring or siblings of centenarians are predisposed to longevity (5). The heritability of human lifespan is still hotly debated, but twin research suggests it is about one third, two-thirds being non-genetic factors (eg, diet, exercise, smoking) (6). The current Special Issue focuses on one such genetic factor – FOXO3 genotype (also known as FOXO3A). The gene FOXO3 is one of only two human genes that has shown consistent replication for association with longevity across multiple human populations. The other is, of course, the apolipoprotein E gene (APOE) (7). These genes might be referred to as the “A list” since no other single gene comes close for relation to human longevity. But with the sheer number of ongoing genetic studies of human aging and longevity this is a rapidly moving target (8).

History of Studies of the Genetics of Aging and Longevity in Humans

The history of genetic studies of human aging and longevity is short. While one could argue about who did what study first, there is a clear progression of studies utilizing centenarians as a model of exceptional human aging and longevity. This began in the 1980s with the Okinawa Centenarian Study (OCS) (9). The OCS was among the first of several large-scale centenarian studies and is the longest continuously running centenarian study in the world. In 1987, the OCS conducted the first candidate gene study of human longevity and discovered that alleles of several HLA genes were positively or negatively associated with longevity, defined as surviving to nonagenarian and/or centenarian years (10). This seminal study led directly to another widely hailed study in the 1990s utilizing similar methodology but comparing genotypes of French and German centenarians with younger aged controls. In this study, Schachter and colleagues were the first to discover the link between alleles of APOE and human longevity (11). This finding was replicated thereafter in multiple populations (7). Despite many subsequent studies, only APOE stood out as a highly replicated “longevity” gene until the discovery of the FOXO3-human longevity link.

Seminal Findings in the Genetics of Aging and Longevity in Model Organisms that led to the First Replication in Humans

In 1988, Friedman and Johnson discovered a recessive mutation in a gene in Caenorhabditis elegans (C. elegans), a nematode model organism widely used in aging research (12). This gene, age-1, was associated with a 40% to 65% increase in lifespan and a significantly reduced mortality rate, compared to controls, suggesting slower aging (13).

The fact that one gene could influence the rate of aging in a model organism of aging was a seminal finding. It led others to explore potential mechanisms and to find aging-related pathways, such as the insulin/IGF-1 genetic pathway (14). This pathway emerged as the first aging-related genetic pathway discovered in any organism. It started with the finding by Kenyon and colleagues in 1993 of a long-lived C. elegans mutant in the gene daf-2 (15). This mutant’s life extension was the longest reported in any organism at that time and required a second gene, daf-16 – the homologue of human FOXO. Morris and colleagues, from Gary Ruvkun’s team, later demonstrated that the C. elegans gene, age-1, encoded a phosphatidylinositol 3-kinase (PI3K) (16). Subsequently, Kimura and colleagues, also from Ruvkun’s team, demonstrated that daf-2 encoded the nematode homolog of the human insulin and IGF-1 receptors that activate downstream PI3K pathways (17). For an excellent review please see Richardson et al (18).

Making the Human Connection

In 2008, a key part this important genetic work in model organisms was replicated in humans. In a groundbreaking study, Willcox, Donlon, Curb and colleagues were the first to report results for any of the human homologs of the several longevity-associated C. elegans genes - or any other model organism genes (19). This team in Hawaii found long-lived Japanese and Okinawan-American men in the Kuakini Honolulu Heart Program, were much more likely to possess specific variants of the human homolog of the long-lived mutant form of daf-16, forkhead/winged helix box 0 subclass, member 3 gene (FOXO3), which encodes the ubiquitously expressed transcription factor, FOXO3. This finding was rapidly replicated in other human studies and is now competing with APOE as the most replicated finding in the genetics of human aging. Importantly, it is the only major longevity-associated gene discovery in humans directly facilitated by model organism work (7).

Over the next 14 years, longevity-associated variants of FOXO3, and genes in its extensive network, have become an intense focus in studies of human aging (20). There is still much to learn, however. Important biological and clinical mechanisms for FOXO3’s protection against aging and deleterious aging-related phenotypes remain undiscovered (21,22). For example, while FOXO3 is highly expressed in the brain, and FOXO3 plays a role in maintaining the neuronal stem cell pool suggesting a potential role in brain aging (23), to our knowledge genome-wide association studies (GWAS) have not yet reported FOXO3 as being strongly associated with brain aging and dementia and very little has been published about FOXO3 in brain health and dementia. So why then is this gene highly expressed in the brain? One finding that has become clear in the literature, is that FOXO3 appears to act, in large part, as a “resilience” gene. In the brain, maintaining the stem cell pool is consistent with keeping the brain more resilient to aging-related pathology, which should protect against cognitive decline. While in other organs/systems, FOXO3 may enhance resilience, and thus slow down the development of other age-related pathology including coronary heart disease (CHD) (24) and cardiometabolic diseases (25) FOXO3 does not appear to prevent such conditions. We hypothesize that it slows down the disease process, such that mortality risk is markedly lowered, equivalent to risk levels of those without such chronic, often life-threatening, conditions. This has important public health implications.

Novel Findings from the Special Issue

The studies presented in this Special Issue address different, but complementary, aspects of recent research on FOXO3 and aging in cells, various model organisms, and humans. Several important and novel findings concerning FOXO3 in aging and aging-related diseases are presented. These include mechanism(s) of action, including new FOXO3 activators and pathways, more support regarding protection against chronic diseases, a new potential role for FOXO3 in preservation of human cognitive function, potential sex-related differences, and public health implications of the longevity-associated genotype. In the present context, “activation” of FOXO3 can involve gene expression as well as mobilization of the protein from the cytoplasm to the nucleus.

One study utilized human cell lines to screen for FOXO3 activators. Jimenez and colleagues (26) screened various health promoting compounds for their FOXO3 activating capacity in different cells, including cancer and renal cell lines. Interestingly, harmine, piperlongumine, and resveratrol activated FOXO3 independently of PI3K/AKT signaling. Harmine’s effect on FOXO3 activity appeared at least partially mediated through the inhibition of dual-specificity tyrosine (Y) phosphorylation regulated kinase 1A (DYRK1A) and showed that the inhibition of sirtuins (SIRTs) reversed this. Activation by these compounds reduced reactive oxygen species and improved chromosomal maintenance 1 (CRM1)-mediated nuclear export. These findings merit further research by examining replication in other cell types. An extensive review of compounds that activate FOXO3 has been published recently (27).

Three studies focused on model organisms of aging. The first study, by Chen and colleagues, utilized Drosophila as a model organism to determine whether zoledronate, a common osteoporosis drug, may act through FOXO3 to improve lifespan and/or healthspan phenotypes (ie, climbing ability, age-related intestinal dysplasia/permeability) (28). Mechanistic studies showed that zoledronate conferred resistance to oxidative stress and reduced accumulation of X-ray-induced DNA damage via inhibition of farnesyl pyrophosphate synthase. The drug was also associated with inhibition of phosphorylated AKT (protein kinase B) in the mammalian target of rapamycin (mTOR) pathway, downstream of the mevalonate pathway, and required dFOXO for its action. This work has important public health implications since zoledronate is widely used to prevent osteoporosis via a yearly dosing schedule and anecdotal evidence in humans suggests that it is associated with better health and longevity.

The second study, by Siswanto and colleagues utilized a C. elegans model and found that chlorogenic acid (CGA) activates nuclear factor erythroid 2-related factor 2 (Nrf2)/skinhead-1 (SKN-1) via the Akt-FOXO3/DAF16a-DNA damage-binding 1 (DDB1) pathway (29). This resulted in prolongation of lifespan in their C. elegans model. Interestingly, the isoform DAF-16a, but not isoform DAF-16f, appeared to regulate the expression of ddb-1 mRNA and SKN-1 protein. CGA increased mean lifespan of daf-16a- and daf-16f-rescued worms by 24% and 9%, respectively. This suggests that both isoforms are important for the lifespan-extending effects of CGA, but that DAF-16a may have a somewhat stronger effect than daf-16f. Thus, Siswanto et al. have established a novel Akt-FOXO3/DAF16a-DDB1 axis that involves a new molecular mechanism for lifespan extension in both nutrient sensing and oxidative stress response pathways in C. elegans.

The third study, by Hao and colleague (30), also used a C. elegans model, with a similar goal. Here they assessed whether a longevity-associated compound, present in the human body, might act through the AMPK/FOXO-dependent pathway. For decades, in a variety of research work, rubidium chloride (RbCl) has been reported to have neuroprotective effects (31). The current researchers hypothesized that rubidium chloride, a trace element in humans, might have therapeutic potential against aging and aging-related diseases in nematodes, and, if so, that this might be translatable to humans. In order to further validate this compound, it is important to understand how it works in the nematode model. If this compound acts through an important, evolutionarily conserved, longevity-associated pathway, then this is more evidence of its potential utility for humans. The current study found: (a) RbCl increases lifespan and enhances stress resistance in C. elegans without affecting fecundity, (b) RbCl induces superoxide dismutase expression, which appears essential for its anti-aging and anti-stress effects, and (c) the AMP-activated protein kinase α subunit AAK-2 and DAF-16 are essential to the anti-aging efficacy of RbCl. RbCl also facilitated translocation of the gene, daf-16, into the nucleus, suggesting the longevity-associated AMPK/FOXO pathway may be important for its beneficial effects in model organisms. The findings have the potential for translatability to humans. Further study of this promising compound is warranted.

Five studies focused on human aging, including two studies of FOXO3 and cognitive heath, one study of FOXO3 and cardiovascular health, another study of FOXO3-linked longevity in rural vs. urban areas, and the last study of sex differences and the potential interaction of SIRT1 and FOXO3 on longevity.

Regarding FOXO3 and cognitive health, Margrett and colleagues investigated interindividual differences in cognitive terminal decline and identified novel determinants, including functional, health, and genetic risk factors, as well as discovering protective factors (32). Data from the Kuakini Honolulu Heart Program/Honolulu-Asia Aging Study, a prospective cohort study of Japanese-American men, were analyzed. The sample was recruited in 1965–1968 when the men were aged 45–68 years. Longitudinal cognitive ability and mortality status were assessed over a span of 20-plus years. Latent class analysis revealed 2 groups: maintainers, who retained relatively high levels of cognitive functioning until death, and decliners, who demonstrated significant cognitive waning several years prior to death. Maintainers were more likely to have greater education, a diagnosis of CHD, higher prevalence of the longevity-associated APOE ε2-allele and FOXO3 G- allele of the single nucleotide polymorphism (SNP) rs2802292. Decliners were more likely to be older and have had a prior stroke, Parkinson’s disease, dementia, greater baseline depressive symptoms, and possess the APOE ε4 risk allele. Significant differences were observed between maintainers and decliners 15 years prior to death, which was much earlier than found by the majority of previous investigations.

A second study of FOXO3 and cognitive health, this by Ji and colleagues, investigated FOXO3, air pollution, and cognitive function in Chinese study participants (33). The study found that the longevity associated FOXO3 genotype may be protective against cognitive impairment.

Since air pollution is a risk factor for cognitive decline and dementia (34), these researchers studied individual and combined effects of FOXO3 genotype and air pollution on cognitive function in a large prospective cohort with up to 14 years of follow-up. Cognitive function was assessed by Mini-Mental State Examination (MMSE). Annual average fine particulate matter (PM2.5) score (ie. air pollution concentrations) were matched within a 1 km² grid. Cross-sectional and longitudinal analyses were conducted using multivariable linear and logistic regression models and generalized estimating equations. At baseline, carriers who were homozygous for minor alleles of longevity-associated FOXO3 SNPs had a higher MMSE score than carriers of homozygous major alleles, which are not associated with longevity. As expected, higher PM2.5 score was associated with a lower MMSE score and higher odds of cognitive impairment. In the longitudinal follow-up, those homozygous for longevity-associated FOXO3 minor alleles had lower probability of cognitive impairment compared with noncarriers. Interestingly, protective effects of the FOXO3 longevity-associated alleles were strongest in older persons, females, and residents in areas with lower air pollution. The finding of stronger cognitive protection amongst residents in rural areas is interesting since one would expect less air pollution in rural areas, and thus less need for FOXO3-related protection. Given higher mortality rates in rural areas from a multitude of biological, psychological and social stressors (eg, smoking, poverty, lack of education, malnutrition, poor job opportunities, long work hours, and inadequate healthcare) the overall stress load is likely much higher in rural China than in urban China – with or without air pollution. The rural setting is precisely the location where the resilient phenotype endowed by FOXO3 longevity-associated genotypes should be most beneficial.

In the third study of humans, addressing FOXO3 and cardiovascular health, Klinpudtan and colleagues performed a cross-sectional study in Japan that included 1,836 older adults in their 70s and 80s (35). They found prevalence of CHD was lower for FOXO3 G-allele carriers in the septuagenarian group for men, and borderline lower in women. After multivariable statistical adjustment for other risk factors, the relation held in men, but reversed in women. These results suggest a potential sex-related difference in risk for prevalent CHD in Japanese men compared with women. To further explore, and possibly confirm, these associations, a longitudinal study with a larger sample size is needed.

The fourth study of humans was of FOXO3-related longevity in rural vs. urban areas. Ji and colleagues conducted a study of FOXO3 and gene-environment interaction in China of 3,085 older adults from the Chinese Longitudinal Healthy Longevity Survey (36).

Given the open cohort design, Cox-proportional hazard regression models were used to assess mortality risk. Longevity-associated minor allele homozygotes of FOXO3 had an apparent protective effect on mortality, while participants living in urban areas had a lower risk of mortality, as expected. The interaction between FOXO3 genotype and urban vs. rural residence was statistically significant. Both higher air pollution (fine particulate matter: PM2.5 score) and lower residential greenness (normalized difference in vegetation index [NDVI]) contributed to higher overall mortality. However, after adjusting for NDVI and PM2.5 score, the size of the effect of the protective longevity-associated alleles of FOXO3 against mortality was only slightly attenuated, suggesting that NDVI and PM2.5 were not major contributors to the mortality difference. Impressively, the effect size of the beneficial FOXO3 alleles on mortality was roughly equivalent to that of living in an urban vs. a rural area. The findings suggest that place of residence and genetic predisposition for longevity may be intertwined.

The fifth and final study of FOXO3 in humans, also by Ji and colleagues, focused on sex differences and the potential interaction of SIRT1 and FOXO3 genotypes on longevity (37). SIRT1 and FOXO3 genetic variants have both been shown to be associated with longevity (38). Molecular biology research in various organisms, such as yeast, C. elegans, and mouse, has demonstrated that SIRT1 has direct and indirect actions on members of the FOXO family of forkhead transcription factor, resulting in improved response to oxidative stress, shifting processes away from cell death toward stress resistance (38). In order to better understand how genotype affects the difference in response to stress by males and females, an open cohort study design of 3,166 community-dwelling participants was conducted in China, by Ji et al. with follow-up from 2008 to 2018. Mean age at baseline was 84.6 years. In 16,375 person-years of follow-up, there were 1,968 deaths. SIRT1 and FOXO3 genotypes exhibited Mendelian randomization as there was no correlation between genes or baseline study population characteristics. Consistent with prior work, particular SIRT1 and FOXO3 alleles demonstrated protective effects on mortality risk. Interestingly, the FOXO3 protective effect was stronger in females, and the SIRT1 protective effect was stronger in males. However, there was no discernable evidence of a synergistic effect on mortality risk for carriers of both SIRT1 and FOXO3 longevity alleles. This is interesting since SIRT1 is upstream of FOXO3 in the insulin/IGF-1 longevity-associated pathway and often considered a “FOXO3 activator” (38). However, how FOXO3 expression is induced is still under scrutiny and traditional genetic pathways appear to be only part of the story for gene-gene communication. Donlon and colleagues demonstrated in an elegant study that FOXO3 acts, at least in part, through a “gene resilience network”, in which neighboring genes physically come together during oxidative and other forms of stress (39). This network is located on chromosome 6 in a “gene neighborhood”, where FOXO3 engages with multiple other genes through actual physical chromatin links, which presumably permit neighboring genes opportunities for activation. While the neighboring genes are not in the FOXO3 transcription pathway they are genes that are relevant to stress responses. It appears that, unlike Vegas, what happens in the FOXO3 gene neighborhood does not stay in the neighborhood.

Conclusion and Future Directions

This Special Issue has brought together research teams from the states of Hawaii, Washington, Oregon, Iowa, Georgia, among others, and diverse continents, from Asia (Japan, including Tokyo, Osaka and Okinawa, and China), Australia, and Europe (including Portugal, Spain and Germany), thus covering a wide swath of the world. The Special Issue articles demonstrate how far-reaching and diverse research on FOXO3 has become. The research teams contributed novel findings on FOXO3’s mechanism(s) of action, its pathways and networks, its contribution to aging, from the cellular level, to the model organism level to the human level. Several teams found novel FOXO3 activators, including potential therapeutic compounds, as well as novel pathways in cells and various model organisms. Other teams contributed novel findings on a potential protective role for FOXO3 in cognitive aging (where very little was known), FOXO3 in cardiovascular disease, FOXO3 in different living environments, FOXO3 in different air quality areas, and found other new protective factors related to FOXO3 genotype.

Important Questions Remain

What are the principal mechanisms of FOXO3’s action? Resilience appears to be a major factor as FOXO3 orchestrates gene expression patterns for response to oxidation, radiation, available energy, protein damage (autophagy), and cellular damage (apoptosis), among other mechanisms.

How does FOXO3 relate to other gene transcription networks implicated in human aging? There are no equitable comparisons at this point, since FOXO3 is in a neighborhood of its own.

What are the clinical implications of FOXO3 genotype? This is a little studied area. Since humans will be more likely to have a longer lifespan with the longevity-associated FOXO3 genotype, will this be accompanied by a longer healthspan? Or a longer deathspan? Of note, in the current Special Issue, the study by Margrett and colleagues (32) demonstrated that FOXO3 longevity allele carriers were more likely to maintain high cognitive function until death compared with non-carriers. This offers a glimmer of hope that FOXO3-related longer lifespan will be associated with a longer healthspan than deathspan.

Future priorities for FOXO3 research may include:

• Identification of additional pathways. Doing so should facilitate targeted therapies at the molecular level.

• Discovery of additional potential FOXO3 activators with minimal side effects so that these can be studied thoroughly.

• Assessment of whether activation of FOXO3 will result in better long-term health outcomes.

• Addressing unanswered questions, such as dose-response effects. Would minimal activation of FOX3O, as seen in hormesis, be optimal? What would be the timing of FOXO3 activation and how would that affect long term health outcomes? Some therapies have positive short-term effects, but negative long-term effects. For example, the anti-inflammatory steroid prednisone is very helpful if used short-term for acute inflammation. But can be quite harmful if used long term, as it may result in accelerated aging phenotypes, such as diabetes and osteoporosis. Would repeated short term FOXO3 activation, such as weekly on/off cycles of a FOXO3 activator, be healthier? Could chronic activation of FOXO3 lead to exhaustion of limited biological resources, such as stem cells?

• Achievement of better “health” in old age (healthspan) – rather than extending lifespan with accompanied by chronic debilitating disease and disability (deathspan).

If we can answer these and other FOXO3-related questions, with a concerted research effort, perhaps we will find out whether Oliver Wendell Holmes was right after all. Maybe you can be 15 years younger than your chronological age.

Box 1 FOXO3 Activation with Aging Intervention Treatment.

Several agents with potential to delay aging are under study (or have been studied) in the National Institute on Aging Interventions Testing Program (ITP), which was designed to find compounds of potential use to optimize human lifespan and healthspan (40). Some promising candidates include: (1) the carotenoid astaxanthin, a strong FOXO3 activator, which has an excellent safety profile; (2) quercetin and (3) fisetin, both are flavonoids that activate FOXO3 and have a reasonable safety profile, with fisetin currently in the ITP but not yet quercetin; the prescription drug (4) metformin, also a FOXO3 activator and (5) another such drug rapamycin, that is an mTOR inhibitor. Rapamycin is not a direct FOXO3 activator but the pathways of each overlap and communicate with each other. These drugs must be approached with more caution if they are to be used to promote healthy aging and longevity in humans. Metform is a first line diabetes drug that increases insulin sensitivity but, in rare cases, can cause severe lactic acidosis. Rapamycin is a chemotherapy adjuvant that makes cancer cells more sensitive to chemotherapy, helps the immune system recover, and decreases cellular growth. It can cause immunosuppression, hypercoagulability, among other mostly reversible side effects. It has been the most successful ITP compound to date, in terms of lifespan and healthspan extension. The latter four are considered senolytics, which are compounds able to remove troublesome senescent cells, and are quite promising, although their efficacy must be balanced with their safety. For a recent reviews of FOXO3 activators and/or senolytics see references (27,41).

Funding

This Editorial work was supported by the Kuakini Medical Center, the US National Institutes of Health (contract N01-AG-4-2149, Grants 5 U01 AG019349-05, 5R01AG027060 [Kuakini Hawaii Lifespan Study], 5R01AG038707 [Kuakini Hawaii Healthspan Study]), 1P20GM125526-01A1 [Kuakini Center of Biomedical Research Excellence for Clinical and Translational Research on Aging], and contract N01-HC-05102 from the National Heart Lung and Blood Institute.

Conflict of Interest

None declared.

Author Contributions

Bradley Willcox, Craig Willcox and Richard Allsopp were the principal editors of this Special Issue, and were responsible for soliciting manuscripts, reviewers, communicating with the corresponding authors, and making final recommendations on manuscripts. Timothy Donlon and Brian Morris greatly contributed to the review of manuscripts. Bradley Willcox, Timothy Donlon, Richard Allsopp and Brian Morris were the principal contributing authors to the Editorial, and all other coauthors read the manuscript and made helpful contributions.