From discoveries in ageing research to therapeutics for healthy ageing

For several decades, understanding ageing and the processes that limit lifespan have challenged biologists. Thirty years ago, the biology of ageing gained unprecedented scientific credibility through the identification of gene variants that extend the lifespan of multicellular model organisms. Here we summarize the milestones that mark this scientific triumph, discuss different ageing pathways and processes, and suggest that ageing research is entering a new era that has unique medical, commercial and societal implications. We argue that this era marks an inflection point, not only in ageing research but also for all biological research that affects the human healthspan.

The past 30 years of ageing research has transitioned from identifying ageing phenotypes to investigating the genetic pathways that underlie these phenotypes. The genetics of ageing research has revealed a complex network of interacting intracellular signalling pathways and higher-order processes 10 . Many of the pathways and processes, such as dietary restriction, that have been identified are known to be critical in homeostatic responses to environmental change. Below, we selected a few key pathways and processes that have emerged during the past 30 years. Insulin-like signalling pathway In 1993, a C. elegans mutation in daf-2 , which is involved in a switch between normal developmental progression and an alternate diapause larval stage (the dauer), was shown to almost double the adult lifespan 11 . This finding was followed by the discovery that two of the daf genes (daf-2 and daf-16) were located in a single pathway that influenced both the formation of the dauer larval stage and adult lifespan 12 . These ageing-associated genes were the C. elegans orthologues of mammalian genes that encode components of the insulin and insulin-like growth factor intracellular signalling pathway (ILS). age-1 turned out to be a phosphatidylinositol-3 kinase, daf-2 encodes an insulin-like receptor and daf-16 encodes a FOXO-like transcription factor that acts downstream of the insulin signalling pathway in mammals. This was supported by findings in yeast 13 and flies 14 , 15 that inhibition of components of the ILS pathway extended lifespan. This suggested that the earlier discoveries in worms were not a ‘private’ mechanism confined to nematodes but a common mechanism with the potential to be relevant to humans and human diseases. Further studies in flies, worms and mice have since proven the conserved effects of inhibiting the insulin signalling pathway and extended lifespan 16 . Some alleles of the daf-16 orthologue in humans, FOXO3 , are also associated with human centenarian populations across the globe 17 , which supports the idea that what we learn from model organisms may be relevant to ageing in humans.

In 1995, a genetic screen identified epigenetic ‘silencing’ factors as longevity genes 24 . Five years later, Sir2 was identified as a conserved protein that regulates replicative lifespan in yeast 25 . A key discovery was the demonstration that Sir2 was a protein deacetylase that removed acetyl groups from histone proteins in a manner that is dependent on the cellular coenzyme nicotinamide adenine dinucleotide (NAD + ) 26 . Another key demonstration was the fact that Sir2 was a key protein in the lifespan extension observed under dietary restriction in yeast 27 . Other organisms also express Sir2-related proteins called sirtuins, which generally function as protein deacylases that remove acyl groups, including acetyl, succinyl and malonyl, from lysine residues on target proteins 28 . Mice and humans express seven sirtuins that are characterized by a conserved catalytic domain and variable N- and C-terminal extensions. SIRT1, SIRT2, SIRT3, SIRT6 and SIRT7 are bona fide protein deacetylases, whereas SIRT4 and SIRT5 do not exhibit deacetylase activity but remove other acyl groups from lysine residues in proteins 29 . Notably, SIRT1, SIRT2, SIRT6 and SIRT7 appear to function as epigenetic regulators, whereas SIRT3, SIRT4 and SIRT5 are located in mitochondria 29 . Sirtuins have emerged as global metabolic regulators that control the response to calorie restriction and protecting against age-associated diseases, thus increasing healthspan and—in some cases—lifespan 30 – 33 . NAD + is a critical redox coenzyme found in all living cells. It serves both as a critical coenzyme for enzymes that fuel reduction-oxidation reactions by carrying electrons from one reaction to another, and as a cosubstrate for other enzymes, such as sirtuins and polyadenosine diphosphate-ribose polymerases (PARPs). There is increasing evidence that NAD + levels and the activity of sirtuins decrease with age and during senescence or in animals on a high-fat diet. By contrast, NAD + levels increase in response to fasting, glucose deprivation, dietary restriction and exercise, which are all conditions associated with a lower energy load 34 – 40 . The fact that NAD + levels increase under conditions that increase lifespan and healthspan, such as dietary restriction and exercise, and decrease during ageing or under conditions that decrease lifespan and healthspan, such as a high-fat diet, support the working model that decreased NAD + levels might contribute to the ageing process. On the basis of this idea, it has been predicted and validated that NAD + supplementation exerts protective effects during ageing 41 , 42 .

In the 1950s, it was theorized that endogenous production of free radical molecules arising from oxygen and generated during fundamental metabolic processes, such as respiration, represent a key factor that drives ageing 48 . These theories particularly focused on mitochondrial production of superoxide as a key mediator of ageing pathophysiology 49 . Indeed, numerous publications have shown that oxidative damage accumulates in multiple tissues and species with age. Although it is indisputable that such damage is one of the most consistent consequences of increasing age in cells and tissues, whether such damage is a cause or a consequence of ageing has proved to be hard to determine. The free radical theory of ageing has proved extremely difficult to test, at least in part because reactive oxygen species are also important signalling molecules. Numerous studies have shown that the modulation of respiration can extend lifespan in model organisms 50 – 53 . In the 1990s and early 2000s, model organisms were used to overexpress key genes involved in detoxifying free radical molecules such as superoxide. There were multiple successes that led to lifespan extension 54 , 55 , which suggests that oxidative damage arising from metabolism was limiting lifespan—at least to some degree. However, this finding was challenged by subsequent studies in mice that showed no increase in the lifespan of wild-type animals 56 in which the key mitochondrial antioxidant protein superoxide dismutase 2 was overexpressed. However, further studies that specifically targeted the hydrogen peroxide scavenger protein catalase to the mitochondria resulted in improved healthspan and increased lifespan in mice 57 – 59 . The contradiction between these two findings in mice suggests that simple genetic overexpression in mammalian systems is very context-specific. This is not surprising, as free radical production within the mitochondria is complex, with at least ten sites of production within the respiratory chain 60 , and the rate of production under diverse physiological states, at various ages and in different cell types remains relatively poorly explored and characterized 61 . Although free radicals are generally implicated in cellular damage and inflammation when present at high levels, they can also potentially increase cellular defenses through an adaptive response (termed ‘mitohormesis’) when present at lower levels 62 . Mitohormesis explains the paradoxical increase in lifespan observed after disruption of mitochondrial function in worms, flies and mice 63 , 64 . Genome-wide screens for lifespan extension in C. elegans revealed that disruption of several genes involved in the electron transport chain extended lifespan 52 , 65 . Inhibition of genes in the mitochondrial electron transport chain triggers the mitochondrial unfolded protein response (UPR mt ), which is also required for the extended lifespan found in these C. elegans mutants 66 . Disruption of mitochondrial function in neurons activates the UPR mt in distal tissues such as the intestine, which suggests the existence of circulating factors that coordinate metabolism between tissues 66 . Mitochondrial perturbation triggered a nuclear transcriptional response that regulates a large set of genes involved in protein folding, antioxidant defenses and metabolism. UPR mt is regulated by several factors including activating transcription factor associated with stress-1 (ATFS-1), the homeobox transcription factor DVE-1, the ubiquitin-like protein UBL-5, the mitochondrial protease ClpP and the inner mitochondrial membrane transporter HAF-1 67 . Studies that demonstrate the importance of mitohormesis in extending lifespan pose several challenges to the field as it is unclear whether using antioxidants would be a good strategy for lifespan extension. As a result, there is evidence both for and against lifespan extension by increasing oxidative stress 63 . It is also unclear how the above findings can be reconciled with results that show that mitochondrial function is enhanced by dietary restriction in multiple species 68 – 70 . Further studies are needed to determine how differing states of mitochondrial function influence ageing in different contexts.

Senescence of the immune system (known as immunosenescence) is one of the causes of ‘inflammaging’, a term coined in 2000 100 that refers to a phenomenon in which older organisms tend to have higher levels of inflammatory markers in their cells and tissues, which results in a low-grade, sterile and chronic pro-inflammatory status. In contrast to acute, transient inflammation—an evolutionarily conserved mechanism designed to protect the host from infections and injuries—inflammaging is linked to a myriad of age-related diseases such as cancer, type 2 diabetes, cardiovascular disease, neurodegenerative diseases and frailty 101 – 105 . Other factors that contribute to inflammaging include genetic susceptibility, obesity, oxidative stress, changes in the permeability of the intestinal barrier associated with translocation of bacterial products (‘leaky gut’), chronic infection and defective immune cells 104 and pro-inflammatory factors that are associated with the senescence-associated secretory phenotype of non-immune senescent cells 76 . In addition, numerous environmental factors—such as the chemicals identified by the Tox21 consortium 106 —can be cytotoxic and pro-inflammatory 101 , 102 . Finally, longevity-enhancing interventions such as dietary restriction reduce inflammatory biomarkers 107 , 108 . On the basis of these findings, inflammaging is now considered to be a biomarker for accelerated ageing and one of hallmarks of ageing biology. As discussed for other variables that influence ageing, an extended lifespan and healthspan may be a result of a fine balance between pro-inflammatory and anti-inflammatory processes 109 . Consistent with this idea, it has been shown that although centenarians have an increased level of pro-inflammatory molecules (for example, interleukin-6, a commonly used marker for chronic morbidity 105 ), the adverse consequences associated with these pro-inflammatory molecules are counterbalanced by high levels of anti-inflammatory molecules 110 .

The rapid increase in our understanding of the molecular mechanisms that underlie ageing has created new opportunities to intervene in the ageing process. Two notable findings have emerged from these early studies. First, the number of genes that can extend lifespan is much larger than expected, which suggests a much higher level of plasticity in the ageing process than expected. Second, genes that control ageing—which define cellular pathways such as the TOR and insulin signalling pathways—are remarkably conserved in yeast, worms, fruit flies and humans. The conservation of these pathways across wide evolutionary distances and the fact that targeting these pathways in model organisms increases both lifespan and healthspan has brought to the fore the idea of interventions in humans. Rapidly ageing societies across the world are seeing an increasing healthcare burden attributable to both morbidity and cost of age-related diseases, such as heart disease, stroke, cancer, neurodegeneration, osteoarthritis and macular degeneration. However, current medical care is highly segmented as well as organ- and disease-based, and ignores the fact that age and the ageing process are the strongest risk factor for each of these diseases. According to the concept of geroscience, targeting conserved ageing pathways is anticipated to protect against multiple diseases and represents a different approach to tackling the rapidly growing burden of diseases worldwide ( Table 1 ). Using geroscience to treat age-related disease The concept of geroscience predicts that conserved ageing pathways are part of the pathophysiology of many age-related conditions and diseases ( Fig. 2 ). For example, multimorbidity is seen as the multisystem expression of an advanced stage of ageing rather than a coincidence of unrelated diseases 117 . Targeting conserved ageing pathways should, therefore, prevent or ameliorate multiple clinical problems. This hypothesis remains to be tested in clinical trials, but is supported by several lines of evidence. A wide range of animal models of specific diseases can be affected by manipulating a single ageing mechanism (such as NAD + ) 118 or senescent cells 95 in the laboratory. Rates of individual age-related diseases and of multimorbidity increase nonlinearly with age, and the rate of acquiring new chronic diseases may be higher in people who have an existing chronic disease 119 . Certain populations, such as people who live with HIV or are homeless, show an early onset of a wide range of age-related chronic diseases and geriatric syndromes that are not necessarily related to their specific disease risks 120 , 121 . A classic statistical analysis of human mortality showed that even curing an entire category of chronic disease, such as all types of cancer or cardiovascular diseases, would only modestly increase life expectancy owing to the expected mortality from other chronic diseases 122 . Conversely, humans with extreme longevity who presumably have favourable ageing mechanisms show delayed onset of most major chronic diseases 123 . In clinical care, multimorbidity is increasingly viewed as an entity unto itself that requires a specific integrative management plan 124 , as intensive but uncoordinated treatment of individual diseases can give rise to the harmful syndrome of polypharmacy 125 . Frailty measures are the most widely used clinical assessments for quantifying the stage of ageing 126 , 127 , and these clinical biomarkers of ageing predict mortality while awaiting liver transplant 128 , complications of surgery 129 and whether the pathology of Alzheimer’s disease manifests as clinical dementia 130 . Taken together, experimental data from preclinical models, epidemiological patterns of age-related conditions and the power of non-disease-specific clinical ageing assessments such as frailty to predict risk and mortality in diverse contexts all suggest that intervening in mechanisms of ageing could have broad clinical benefit.

Both drugs that are being developed to target ageing and some drugs that are commonly used behave as geroprotectors in animal models. The multicentre Intervention Testing Program (ITP), which is supported by the National Institute for Ageing, has identified five drugs that reproducibly increase lifespan in genetically heterogenous mice 147 , including rapamycin, acarbose, nordihydroguaiaretic acid, 17-α-oestradiol and aspirin 147 . Some of these drugs also improve healthspan measures in some tissues of animal models 148 – 150 . Drugs found in other studies to extend rodent lifespan include metformin 151 (although it did not repeat in the ITP at the same dose), drugs targeting the angiotensin-converting enzyme and the aldosterone receptor, and the sirtuin activators SRT2104 152 and SRT1720 153 . Further work will be necessary to validate that these drugs act as true geroprotectors in model organisms. A key question is how these interventions will be tested and eventually used clinically in humans. Geroscience predicts that ageing therapies will ameliorate or prevent several age-related diseases and conditions simultaneously. Clinical trials to test this hypothesis should therefore use clinical outcomes that inherently depend on multiple age-related diseases or conditions. Examples include multimorbidity, or the combination of several age-related chronic diseases; the multifactorial geriatric syndromes such as frailty or delirium; or resilience to health stressors such as surgery or infection 154 . Multimorbidity and frailty are also widely incorporated into measures of age-related risk that inform clinical decision-making 155 . Other examples of such measures that could be useful trial outcomes include grip strength, gait speed 156 , timed-up-and-go and activities of daily living. These clinical measures of ageing could be useful for selecting patients at higher age-related risk to receive interventions. For example, the risk of multimorbidity increases steeply with age 119 ; however, developing one chronic disease increases the risk of developing another by several fold 157 . How early or late in the ageing process interventions can be effective remains to be seen, although animal studies of drugs and human studies of exercise provide some reassurance that the window of opportunity extends quite late in life. At least five major classes of drugs are currently being tested in humans for their geroprotective potential. Metformin. Metformin is a widely prescribed antidiabetic drug that has been found to target several molecular mechanisms of ageing 158 . A retrospective analysis of patients with diabetes who received metformin showed increased lifespan in comparison to individuals without diabetes 159 . In randomized trials, metformin prevented the onset of diabetes, improved cardiovascular risk factors and reduced mortality 160 , 161 . Epidemiological studies have suggested that metformin use might also reduce the incidence of cancer and neurodegenerative disease 158 . These data underpin the proposed Targeting Aging with MEtformin (TAME) study, a large randomized controlled trial of metformin given to 65- to 80-year-old individuals without diabetes who are at high risk for the development of chronic diseases of ageing. The primary outcome of TAME is a composite of death or new major age-related chronic diseases, including cardiovascular disease, cancer and dementia. Other outcomes include geriatric measures such as mobility, independence in activities of daily living and cognitive function 162 .

As discussed above, senolytic drugs—drugs that selectively target and eliminate senescent cells—have showed great geroprotective potential in animal models 95 . Some of these drugs are natural products 96 , 97 , whereas others are synthetic small molecules 90 , 98 , 99 . A growing number of biotechnology companies and research laboratories are developing new or repurposed senolytics that have just begun to be tested for safety in humans, with no results—thus far—regarding efficacy.

NAD + precursors such as nicotinamide riboside and nicotinamide mononucleotide aim to supplement the age-associated decrease in cellular NAD levels 69 . In animal models, both precursors have shown geroprotective activity against a number of ageing-associated diseases. Several companies are currently selling nicotinamide riboside and nicotinamide mononucleotide as supplements online. Although these supplements increase NAD levels in humans 170 , no efficacy or geroprotective effects for humans have been demonstrated thus far.

Diet is probably one of the most important influences on health and ageing. However, it is an enormously complicated topic and beyond the scope of this Review (extensive discussion on this topic has previously been published 176 ). The field of ageing has focused almost exclusively on the lifespan and healthspan effects of dietary restriction but, at the other end of the spectrum, overeating and the accompanying obesity shortens lifespan and decreases healthspan. In between these two extremes, there is strong evidence that optimal eating is associated with increased life expectancy and a reduction in the risk of all types of chronic disease. Many claims have been made for the competitive merits of different diets relative to one another. However, it is very difficult to conduct rigorous, long-term studies that compare different diets for their lifespan and healthspan effects using methodology that is devoid of bias and confounding variables. Without such direct comparisons, no specific diet can claim superiority over any others. However, a number of themes have emerged from studies that compare different diets and from the study of populations that are geographically associated with increased longevity (so-called ‘blue zones’). Diets that favour longevity and healthspan are generally characterized by minimally processed foods, being predominantly plant-based, low alcohol consumption and a lack of overeating. Exciting recent developments are emerging in the nutrition field, such as intermittent fasting 177 , diets that mimic fasting 178 and time-restricted feeding 179 . Recently, interest has grown in a ketogenic diet that is characterized by the endogenous production of high levels of the ketone body β-hydroxybutyrate. This diet has long been used as a treatment for childhood epilepsy and was recently shown to increase healthspan in two separate studies in mice 180 – 182 . The studies are supported by recent findings that β-hydroxybutyrate modulates the enzymatic activity of the epigenetic regulators, histone deacetylases, and thereby activates the expression of FOXO3 183 . Future research will focus on the healthspan and possible lifespan of these dietary interventions and the identification of their interactions with pathways that regulate ageing.