DNA damage theory of aging and sex

DNA damage theory of aging and sex

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If aging is due to accumulation of damages in DNA, then shouldn't the DNA in gametes also be damaged and so the next generation should be borne old! How does gametes avoid this DNA damage accumulation?

I've answered a related question, which was wondering why older people get more cancer from accumulated DNA damage and yet their children aren't immediately susceptible to it, here:

Quoting from my answer there:

… a parent does not pass on all their mutations in all their cells, they pass on just one, which need not have any deleterious mutations at all, even if the parent has cancer themselves in some other part of the body.

If that one cell is defective in some way, it is less likely to be involved in creation of a successful pregnancy, and may lead to miscarriage. Therefore, the probabilistic process of reproduction tends to result in offspring from selection of healthier gametes.

There are in fact mutations that accumulate in gametes, but if any of those mutations are very serious, they are unlikely to support development of an entire new organism, and they are also relatively few. The burden of age on the genome is spread over all the cells in a body, and worst in types of cells that divide the most, when errors are most likely to occur.

Yes and no. Your concept about DNA damage causing aging is good. But aging is caused by DNA damage that causes the biologic / signaling pathways to not work as well as they once did. Think of biophysics and interactions among the plethora of proteins, molecules, etc. in your cells and tissues and all of the interactions therewith where even the electronic charge + vs - and quantity of molecules (charged or not) in a vicinity influences the effectiveness of a biological pathway etc. This is aging. Basically, if you have learned about development biology at the molecular and cellular levels, you would know that much of that is not yet developed at the gamete stage. Therefore, there's not a whole lot of aging that can be had at the gamete stage because there's frankly nothing going on just DNA damage. Granted the DNA damage can influence on a grander scale as Bryan points out above - life or death (rather non-life) or the imposition of birth defects and other serious issues - but this is not how we define aging even if they are somewhat conceptually related. In short, aging is caused by the inefficiency and impairment of otherwise optimal (at birth) biological processes that slowly decay over time (silent mutations, environmental impositions, etc. that cause biophysics-based alterations at the smallest scale that impact exponentially as magnified over multiples of molecular interactions across a grand array of biological pathways and other molecular/cellular interactions).

A basic fact of male biology may be responsible for men's short lifespans

Why do males typically have shorter lives than females?

That’s the question that evolutionary biologist Doris Bachtrog has been exploring in her lab at the University of California, Berkeley. And finally, she’s encountered an answer: A new study on flies from Brachtrog’s lab suggests the repeating DNA found in the Y-chromosome might be responsible for males’ shorter lifespan.

This finding was published Thursday in PLOS Genetics.

Let’s back up — Inside human cells are strings of DNA (deoxyribonucleic acid). Genes are a specific part of DNA they give instructions to cells to make certain proteins. The remaining DNA is called heterochromatin. Heterochromatin is very densely packed DNA and has a variety of functions. Some heterochromatin helps regulate genes, while other heterochromatin protects the integrity of chromosomes. Bachtrog and colleagues are interested in why chromosomes can, in turn, become toxic.

It’s established that, in every animal species where there is a lifespan difference between sexes, the sex with the equivalent of the Y chromosome has a shorter lifespan. One theory for why this is has to do with repeating sequences of DNA. Both sexes have repeating sequences of DNA, but the Y chromosome has significantly more than the X chromosome.

“XX females have some heterochromatin, but much less so than XY males, since the number of repeats on the Y chromosomes is much much larger than on the X,” Bachtrog tells Inverse.

This theory is called the “toxic Y effect.”

What’s new — In the study, Bachtrog and colleagues wanted to test for an association between sexes with heteromorphic (different from each other, so XY as opposed to XX) sex chromosomes, sex-specific heterochromatin, and repetitive DNA sequences.

Specifically, the team wanted to examine transposable elements (TE). These are repeated sequences of DNA that are constrained or “silenced” by that dense packing but — if that tense packing is removed — have the potential to move from one genomic location to another.

A useful species for this line of study are fruit flies which, according to the study authors, have “well-established” Y-chromosomes and a notoriously short lifespan. Also, the male fruit flies have about twice as much repeating DNA as the females.

The researchers found that when male fruit flies are young, the repeat DNA is packed so tightly that the repeat sections of DNA are effectively turned off.

As the fly aged, however, these repetitious sections became less tightly packed together. This, in turn, can lead to the repeated DNA sections being transcribed — that’s when cells following the directions to create proteins. And once they’re out of those densely packed constraints, there’s the possibility that they can move around to other genomic locations.

Bachtrog explains that transposable elements will “cut themselves out of particular regions of the genome [causing DNA damage], and insert into a new region [and can cause genes to become inactive if they insert into a gene].”

Why this matters — Given that previous studies suggest that when this repeat DNA is activated fruit flies experience difficulties like memory loss and DNA damage, the study results make the case for more research into repeat DNA and heterochromatin.

While fruit flies are clearly not a perfect analog for humans — for one thing, a fruit fly’s Y chromosome is far more repetitive than a human’s — in this case, there are useful similarities.

“Humans contain large stretches of repetitive DNA, and heterochromatin alterations have been identified as drivers of human aging,” Bachtrog says. “Like in flies, human males tend to live shorter than females, suggesting that differences in heterochromatin content may contribute to sex-specific aging in our species as well.”

Next, Bachtrog says she’s planning to use the fruit fly model developed in this study to research “the molecular basis of heterochromatin loss, and sex-specific differences.”

Plenty of other things — including diet, exercise, and even the strength of our social connections — also contribute to how long we live. But understanding how our DNA also contributes to longevity is still important. It helps explains a mysterious element of life on a fundamental level. It also points to how these assumed side-effects of being alive can one day be manipulated, especially with technologies like CRISPR potentially on the horizon.

DNA damage theory of aging and sex - Biology

Why do we age? There are many theories that attempt to explain how we age, however, researchers still do not fully understand what factors contribute to the human lifespan (Jin, 2010). Research on aging is constantly evolving and includes a variety of studies involving genetics, biochemistry, animal models, and human longitudinal studies (NIA, 2011a). According to Jin (2010), modern biological theories of human aging involve two categories. The first is Programmed Theories that follow a biological timetable, possibly a continuation of childhood development. This timetable would depend on “changes in gene expression that affect the systems responsible for maintenance, repair, and defense responses,” (p. 72). The second category includes Damage or Error Theories which emphasize environmental factors that cause cumulative damage in organisms. Examples from each of these categories will be discussed.

Genetics: One’s genetic make-up certainly plays a role in longevity, but scientists are still attempting to identify which genes are responsible. Based on animal models, some genes promote longer life, while other genes limit longevity. Specifically, longevity may be due to genes that better equip someone to survive a disease. For others, some genes may accelerate the rate of aging, while others decrease the rate. To help determine which genes promote longevity and how they operate, researchers scan the entire genome and compare genetic variants in those who live longer with those who have an average or shorter lifespan. For example, a National Institutes of Health study identified genes possibly associated with blood fat levels and cholesterol, both risk factors for coronary disease and early death (NIA, 2011a). Researchers believe that it is most likely a combination of many genes that affect the rate of aging.

Evolutionary Theory: Evolutionary psychology emphasizes the importance of natural selection that is, those genes that allow one to survive and reproduce will be more likely to be transmitted to offspring. Genes associated with aging, such as Alzheimer Disease, do not appear until after the individual has passed their main reproductive years. Consequently, natural selection has not eliminated these damaging disorders from the gene pool. If these detrimental disorders occurred earlier in the development cycle, they may have been eliminated already (Gems, 2014).

Figure 9.10 Telomeres and Cellular SenescenceAdapted from National Institute on Aging

Cellular Clock Theory: This theory suggests that biological aging is due to the fact that normal cells cannot divide indefinitely. This is known as the Hayflick limit, and is evidenced in cells studied in test tubes, which divide about 40-60 times before they stop (Bartlett, 2014). But what is the mechanism behind this cellular senescence? At the end of each chromosomal strand is a sequence of DNA that does not code for any particular protein, but protects the rest of the chromosome, which is called a telomere. With each replication, the telomere gets shorter. Once it becomes too short the cell does one of three things. It can stop replicating by turning itself off, called cellular senescence. It can stop replicating by dying, called apoptosis. Or, as in the development of cancer, it can continue to divide and become abnormal. Senescent cells can also create problems. While they may be turned off, they are not dead, thus they still interact with other cells in the body and can lead to an increase risk of disease. When we are young, senescent cells may reduce our risk of serious diseases such as cancer, but as we age they increase our risk of such problems (NIA, 2011a). Understanding why cellular senescence changes from being beneficial to being detrimental is still under investigation. The answer may lead to some important clues about the aging process.

DNA Damage: Over time DNA, which contains the genetic code for all organisms, accumulates damage. This is usually not a concern as our cells are capable of repairing damage throughout our life. Further, some damage is harmless. However, some damage cannot be repaired and remains in our DNA. Scientists believe that this damage, and the body’s inability to fix itself, is an important part of aging (NIA, 2011a). As DNA damage accumulates with increasing age, it can cause cells to deteriorate and malfunction (Jin, 2010). Factors that can damage DNA include ultraviolet radiation, cigarette smoking, and exposure to hydrocarbons, such as auto exhaust and coal (Dollemore, 2006).

Mitochondrial Damage: Damage to mitochondrial DNA can lead to a decaying of the mitochondria, which is a cell organelle that uses oxygen to produce energy from food. The mitochondria convert oxygen to adenosine triphosphate (ATP) which provides the energy for the cell. When damaged, mitochondria become less efficient and generate less energy for the cell and can lead to cellular death (NIA, 2011a).

Figure 9.11 Free Radicals

Free Radicals: When the mitochondria uses oxygen to produce energy, they also produce potentially harmful byproducts called oxygen free radicals (NIA, 2011a). The free radicals are missing an electron and create instability in surrounding molecules by taking electrons from them.

There is a snowball effect (A takes from B and then B takes from C, etc.) that creates more free radicals which disrupt the cell and causes it to behave abnormally (See Figure 9.11). Some free radicals are helpful as they can destroy bacteria and other harmful organisms, but for the most part they cause damage in our cells and tissue. Free radicals are identified with disorders seen in those of advanced age, including cancer, atherosclerosis, cataracts, and neurodegeneration. Some research has supported adding antioxidants to our diets to counter the effects of free radical damage because the antioxidants can donate an electron that can neutralize damaged molecules. However, the research on the effectiveness of antioxidants is not conclusive (Harvard School of Public Health, 2016).

Figure 9.12 President Obama

Immune and Hormonal Stress Theories: Ever notice how quickly U.S. presidents seem to age? Before and after photos reveal how stress can play a role in the aging process. When gerontologists study stress, they are not just considering major life events, such as unemployment, death of a loved one, or the birth of a child. They are also including metabolic stress, the life sustaining activities of the body, such as circulating the blood, eliminating waste, controlling body temperature, and neuronal firing in the brain. In other words, all the activities that keep the body alive also create biological stress.

To understand how this stress affects aging, researchers note that both problems with the innate and adaptive immune system play a key role. The innate immune system is made up of the skin, mucous membranes, cough reflex, stomach acid, and specialized cells that alert the body of an impending threat. With age these cells lose their ability to communicate as effectively, making it harder for the body to mobilize its defenses. The adaptive immune system includes the tonsils, spleen, bone marrow, thymus, circulatory system and the lymphatic system that work to produce and transport T cells. T-cells, or lymphocytes, fight bacteria, viruses, and other foreign threats to the body. T-cells are in a “naïve” state before they are programmed to fight an invader, and become “memory cells”. These cells now remember how to fight a certain infection should the body ever come across this invader again. Memory cells can remain in your body for many decades, and why the measles vaccine you received as a child is still protecting you from this virus today. As older adults produce fewer new T-cells to be programmed, they are less able to fight off new threats and new vaccines work less effectively. The reason why the shingles vaccine works well with older adults is because they already have some existing memory cells against the varicella virus. The shingles vaccine is acting as a booster (NIA, 2011a).

Hormonal Stress Theory, also known as Neuroendocrine Theory of Aging, suggests that as we age the ability of the hypothalamus to regulate hormones in the body begins to decline leading to metabolic problems (American Federation of Aging Research (AFAR) 2011). This decline is linked to excess of the stress hormone cortisol. While many of the body’s hormones decrease with age, cortisol does not (NIH, 2014a). The more stress we experience, the more cortisol released, and the more hypothalamic damage that occurs. Changes in hormones have been linked to several metabolic and hormone related problems that increase with age, such as diabetes (AFAR, 2011), thyroid problems (NIH, 2013), osteoporosis, and orthostatic hypotension (NIH, 2014a).

DNA Damage Responses in Atherosclerosis

Kenichi Shimada , . Moshe Arditi , in Biological DNA Sensor , 2014

DNA damage exists in all cellular organisms. While DNA damage is distinguished from mutation, mutation can result from unrepaired DNA. While most DNA damage can be repaired, such repair systems are not 100% efficient. Un-repaired DNA damage accumulates in non-replicating cells, such as neurons or myocytes of adult mammals, and can cause aging. DNA damage can be subdivided into two types: (1) endogenous damage caused by reactive oxygen species (ROS) that are derived from metabolic byproducts and (2) exogenous damage caused by radiation (UV, X-ray, gamma), hydrolysis, plant toxins, and viruses. Current data suggest that increased oxidative stress is a major characteristic of hypercholesterolemia-induced atherosclerosis and that oxidative stress is most likely associated with DNA damage in the development of cholesterol-induced plaques. This chapter critically addresses the extent to which the DNA damage, the sensing of it, and DNA damage repair are involved in the pathogenesis of atherosclerosis.

Genes and Bodily Functions

Before discussing the key concepts related to aging and genetics, let's review what our DNA is and some of the basic ways in which genes affect our lifespan.

Our genes are contained in our DNA which is present in the nucleus (inner area) of each cell in our bodies. (There is also mitochondrial DNA present in the organelles called mitochondria which are present in the cytoplasm of the cell.) We each have 46 chromosomes making up our DNA, 23 of which come from our mothers and 23 which come from our fathers. Of these, 44 are autosomes, and two are the sex chromosomes, which determine if we are to be male or female. (Mitochondrial DNA, in contrast, carries much less genetic information and is received from only our mothers.)

Within these chromosomes lie our genes, our genetic blueprint responsible for carrying the information for every process which will take place in our cells. Our genes can be envisioned as a series of letters that make up words and sentences of instructions. These words and sentences code for the manufacturing of proteins that control every cellular process.

If any of these genes are damaged, for example, by a mutation that alters the series of "letters and words" in the instructions, an abnormal protein may be manufactured, which in turn, performs a defective function. If a mutation occurs in proteins that regulate the growth of a cell, cancer may result. If these genes are mutated from birth, various hereditary syndromes may occur.   For example, cystic fibrosis is a condition in which a child inherits two mutated genes controlling a protein that regulates channels responsible for the movement of chloride across cells in the sweat glands, digestive glands, and more. The result of this single mutation results in a thickening of mucus produced by these glands, and the resultant problems which are associated with this condition.  

2 - The biology of ageing

This chapter introduces key biological concepts of ageing. First, it defines ageing and presents the main features of human ageing, followed by a consideration of evolutionary models of ageing. Causes of variation in ageing (genetic and dietary) are reviewed, before examining biological theories of the causes of ageing.

Thanks to technological progress in different areas, including biomedical breakthroughs in preventing and treating infectious diseases, longevity has been increasing dramatically for decades. The life expectancy at birth in the UK for boys and girls rose, respectively, from 45 and 49 years in 1901 to 75 and 80 in 1999 with similar figures reported for other industrialized nations (see Chapter 1 for further discussion). A direct consequence is a steady increase in the proportion of people living to an age where their health and well-being are restricted by ageing. By the year 2050, it is estimated that the percentage of people in the UK over the age of 65 will rise to over 25 per cent, compared to 14 per cent in 2004 (Smith, 2004).

The greying of the population, discussed elsewhere (see Chapter 1), implies major medical and societal changes. Although ageing is no longer considered by health professionals as a direct cause of death (Hayflick, 1994), the major killers in industrialized nations are now age-related diseases like cancer, diseases of the heart and neurodegenerative diseases.

The Cycle of Growth and Replication

The growth and replication of cells is often described as a cyclic process with two main phases: interphase , when the cell grows and replicates DNA in preparation for cell division, and mitosis , during which the actual division of the cell into two daughter cells occurs. The events occurring in this cyclic process are summarized in the diagram on the right in which interphase events are shown with blue arrows and mitosis is shown in brown. Note that cells may also exit the cycle and enter a G0 phase either temporarily or more or less permanently. In cells that are actively growing and dividing, such as those in an embryo, the cycle is completed frequently as cells divide over and over as the embryo grows and develops. In adults the need for growth and development has passed, and most cells remain in the G0 phase during which they perform their specialized functions, but they no longer replicate (e.g., nerve and muscle cells). Nevertheless, even in fully developed adults certain progenitor cells retain the ability to replicate and give rise to new daughter cells to replace cells that are damaged or lost due to wear and tear. For example, Clara cells in the epithelium of the respiratory tract have the capacity to replicate to produce two daughter cells - one that will be a new Clara cell and one that will differentiate into a replacement epithelial cell. Similarly, hematopoetic stem cells in bone marrow have the ability to replicate to give rise to progenitor cells that can differentiate into the various cellular elements of blood.

Regulation of the cell cycle is of critical importance to the aging process. Replication should only occur when there is a need for growth and development (in embryos and the young) or when there is a need to replace damaged or lost cells. Thus, the cycle is influenced by growth factors and by proto-oncogenes that favor replication and by anti-oncogenes that produce proteins that inhibit replication. These various factors interact to regulate the cell cycle in cells that have retained the capacity to divide. In addition, there are cellular processes that constitute checkpoints that prevent the cell cycle from proceeding if errors have occurred. The first checkpoint occurs during the G1 phase and provides an opportunity for cellular that processes to repair damaged DNA before the cell enters the S phase when replication occurs. This prevents the daughter cells from inheriting damaged DNA, which would result in mutations. There are also other checkpoints in S and G2 phases that check for damaged DNA and failure of DNA replication. The final cell cycle checkpoint occurs at the end of mitosis and checks for any chromosomes that have been misaligned. The many factors that regulate the cell cycle play an important rol in the aging process, because as cells age their capacity to replicate diminishes to the point that they are no longer able to divide. As this occurs, the ability to replace damaged or lost cells dwindles and ultimately results in declines in tissue strength and cellular and organ function that are characteristic of aging.


Ageing appears to be a nearly universal feature of life, ranging from unicellular microorganisms to humans. Longevity depends on the maintenance of cellular functionality, and an organism's ability to respond to stress has been linked to functional maintenance and longevity. Stress response pathways might indeed become therapeutic targets of therapies aimed at extending the healthy lifespan. Various progeroid syndromes have been linked to genome instability, indicating an important causal role of DNA damage accumulation in the ageing process and the development of age-related pathologies. Recently, non-cell-autonomous mechanisms including the systemic consequences of cellular senescence have been implicated in regulating organismal ageing. We discuss here the role of cellular and systemic mechanisms of ageing and their role in ageing-associated diseases.

1. Introduction

Ageing can be defined as a state of progressive functional decline accompanied by an exponential increase in mortality (the Gompertz law [1–3]). Despite being widespread among almost all multicellular organisms [4,5], there are exceptions. The existence of species without an observable time-dependent functional decline and increase in mortality, termed ‘negligible senescence’ [6–8], suggests that the ageing process is not an entirely ubiquitous, inevitable one, hence raising the important questions of ‘why does it happen?’ and ‘how can it be so variable?’

In the wild, extrinsic factors are the ones mostly leading to mortality: animals tend not to grow very old and, as a result, the power of natural selection declines over time. Natural selection is, therefore, predicted to only have a weak influence on the process of senescence, making the existence of genes that actively promote ageing very unlikely [9]. Instead, according to the ‘mutation accumulation’ theory, this lack of selective effects in later life stages allows the accumulation of alleles with late, unselected effects over several generations [9]. Alternatively, the ‘antagonistic pleiotropy’ theory proposes a major contribution of pleiotropic genes—genes selected to maintain fitness during early life but with unselected deleterious effects later, after the organism's reproductive period—in the development of age-related phenotypes [4]. Finally, the ‘disposable soma’ theory states that, because of resource scarcity, organisms evolved mechanisms to optimally allocate metabolic resources into reproduction at the expense of somatic maintenance. Proper somatic maintenance is only required to ensure that an organism reaches reproductive maturity therefore, it can be beneficial not to invest resources into somatic repair and maintenance even if that will lead to damage accumulation over time, ultimately driving the ageing process [5,10]. Both the ‘antagonistic pleiotropy’ and ‘disposable soma’ theories are based on the idea of a cellular ‘trade-off’—a compromise, where mechanisms that are at first advantageous bring detrimental consequences later on.

In this review, we primarily focus on the role of DNA damage accumulation in pathology and in the ageing process. We start by highlighting evolutionary trade-offs between somatic maintenance and reproduction and how these can be tightly connected to an organism's environment we then move on to the role of DNA repair pathways in enforcing these trade-offs at the cellular and organismal level. Finally, we give special attention to non-cell-autonomous DNA damage responses (DDRs), which promote tissue dysfunction and compensatory responses with the aim of re-establishing tissue homeostasis, as the study of these will surely facilitate the identification of the mechanisms underlying the systemic effects of DNA damage in the future.

2. Influence of the environment on lifespan and ageing

Seeing that organisms inhabit highly variable environments, it is not surprising that there is a vast range of highly specialized traits and responses that promote survival and/or optimal reproduction for those specific environments and circumstances. In the nematode Caenorhabditis elegans, some of these environmental responses are very well characterized. Temperature is a major influencing factor in the nematode's lifespan: hermaphrodite worms have a half-life of about 13 days at 20°C but their lifespan can be modulated by the environment's temperature [11]. A decrease in temperature to 16°C increases the worms' half-life to approximately 20 days, while an increase in temperature to 25°C has the opposite effect, decreasing half-life to approximately 8 days [11].

In addition to temperature, food availability is also a major modulator of the worms' lifespan. When faced with starvation, C. elegans can enter diapause—a physiological state of dormancy and developmental delay, with halted feeding and reproduction [12–14]. Depending on the developmental stage at which the worms face starvation, distinct diapause states can be established. Dauer arrest, the most well-studied diapause state, is established when worms are starved at the L2 larval stage. Dauer worms undergo specific anatomical and metabolic modifications and are surprisingly resistant to different environmental stressors when compared with non-dauer worms [14–16]. Importantly, worms have been reported to survive up to several months in this stage but are still able to resume development, reach adulthood and display normal adult lifespan and reproduction when faced again with ideal conditions [14]. A distinct diapause state—adult reproductive diapause (ARD)—is established under conditions of starvation and high larval density shortly after the transition from the L4 larval stage into young adulthood [17]. Unlike dauer, this state is not associated with major anatomical changes and, while in this state, worms show some signs of tissue and cellular ageing, including atrophy of the intestine and germline degradation [17]. Remarkably, shortly after exiting ARD, worms display normal adult morphology (including a repopulated germline and functional intestine) and lifespan [17]. This rejuvenation process becomes even more extraordinary because it takes place in adult worms, in which all somatic cells are postmitotic, strongly hinting towards the existence of signalling pathways promoting tissue functionality or, in this case, rejuvenation in a systemic way following stressful conditions. The mediators involved in this rejuvenation process are still unknown however, reactivation of RNA metabolism appears to be a requirement for somatic restoration post-ARD to occur [18].

The impact of food availability in stress responses and longevity is not restricted to C. elegans. The effects of caloric restriction (CR) (reduction in caloric intake without malnutrition) in slowing the ageing process and promoting health have been extensively described in animal models and recent studies have suggested that this might translate even to human health maintenance [19–21]. CR is hypothesized to trigger an evolutionary conserved adaptive response for periods of food scarcity responsible for shifting an organism's energy resources from growth and reproduction to somatic maintenance [22,23]. The trade-off becomes particularly apparent in C. elegans, where extraordinarily long-lived dauer larvae maintain somatic function until they resume offspring generation once food becomes available and are then subject to age-related somatic decline. According to the disposable soma theory, it is not surprising to observe a correlation between longevity and the amount of resources applied to somatic maintenance and repair. This is well supported by classical studies reporting a correlation between DNA repair capacity and mammalian lifespan [24,25].

3. The DNA damage response

One important aspect of the ageing process is the accumulation of DNA damage through time [26,27]. While containing the entire genetic information (except for mitochondria-encoded genes), the nuclear genome is constantly threatened by genotoxic insults, with an estimated frequency of the order of tens of thousands per day [28]. These hazards can arise from exogenous or endogenous sources. Exogenous sources are, to some extent, avoidable these include ultraviolet (UV) and ionizing radiation and a variety of genotoxic chemicals. Endogenous sources, on the other hand, are unavoidable as they include metabolic by-products, such as reactive oxygen species (ROS), and spontaneous chemical reactions that target DNA molecules (including alkylation and hydrolysis of DNA chemical bonds) [28,29]. The lesion type inflicted on the DNA greatly depends on the source of the damage. Lesions caused by endogenous sources tend to arise stochastically at a higher rate. Single-strand breaks (SSBs) constitute the majority of DNA lesions, as they can arise from base hydrolysis and oxidative damage [30]. Stochastic errors during DNA replication occur at a low rate but may lead to single-nucleotide substitutions and ROS cause oxidative DNA lesions such as 8-oxoguanine [31]. Lesions caused by exogenous sources can be mutagenic and also highly cytotoxic. For instance, exposure to UV radiation leads to helix-distorting lesions such as 6–4 photoproducts [32] and, most predominantly, cyclobutane pyrimidine dimers [33] chemotherapeutic interventions can also induce interstrand cross-links (ICLs) and double-strand breaks (DSBs) [34,35] (figure 1).

Figure 1. Different types of DNA lesions and corresponding DNA repair systems. Distinct DNA lesions have distinct consequences for a cell. Nucleotide substitutions followed by misreplication lead to accumulation of mutations and chromosomal aberrations, increasing the risk of cancer development. By contrast, bulkier lesions can also block replication and transcription, leading to cell-cycle arrest and, possibly, cell senescence or apoptosis. To avoid this, cells have evolved complex, highly conserved DNA repair systems capable of responding to specific types of lesions. Base mispairs (1) and short deletions/insertions are repaired by mismatch repair (MMR). Single-strand breaks (2) are repaired by complex SBBR cascades. Helix-distorting lesions, such as cyclobutane pyrimidine dimers (3), are repaired by the nucleotide excision repair (NER) pathway. Oxidative lesions and small alkylation products (4) are repaired by base excision repair (BER). Highly cytotoxic double-strand breaks (5) are either repaired by the efficient but error-prone non-homologous end-joining (NHEJ) pathway or by the precise homologous recombination (HR) pathway.

DNA damage can have distinctive consequences for cells. Persistent nucleotide substitutions, due to erroneous repair followed by misreplication, lead to the accumulation of permanent mutations and chromosomal aberrations, which increase the risk of cancer development [36]. By contrast, bulky types of DNA lesions can block transcription and replication, triggering the arrest of the normal cell cycle, ultimately leading to cell senescence or cell death, both states preventing the cell from transforming into tumour cells but ultimately contributing to ageing [36]. Nuclear DNA requires constant maintenance to be kept intact and error-free in order to avoid the aforementioned consequences. For this, cells evolved intricate, evolutionarily highly conserved machineries mediating cellular responses to DNA damage—termed the ‘DDR’. These highly complex systems include not only several repair pathways specific for different types of lesion but also distinct signalling cascades of damage sensors, signal boosters and effectors responsible for deciding the cell's fate. This system has two immediate goals: (i) arrest the cell cycle to prevent the propagation of corrupted genetic information, while providing time to repair the damage, and (ii) actually coordinate the repair of the DNA lesion. Depending on the success of these previous steps, the cell's fate is then decided: after lesions are successfully repaired, the DDR signalling ceases, cells survive and return to their original state however, impossible to repair lesions trigger a persistent DDR signalling which can then engender cellular senescence or apoptosis [37,38]. Given the harmful consequences of irreparable DNA damage, it is not surprising that defects in DNA repair pathways are associated with severe human pathological conditions.

4. Genome instability syndromes

Human genome instability syndromes support the link between genome stability and human health, particularly premature ageing and cancer. These syndromes are typically characterized by chromosomal instability and hypersensitivity to DNA-damaging agents, thus increasing cancer predisposition and exacerbating the progressive degeneration of specific tissues [36,39–41]. Owing to the large variety of DNA lesions, cells evolved specialized, lesion-specific repair systems. Defects in these repair systems can, however, have highly distinct functional consequences.

The most common DNA lesions, SSBs, are repaired by complex single-strand break repair (SBBR) signalling cascades initiated when the sensor protein poly(ADP-ribose) polymerase 1 (PARP1) detects and binds to SBBs [30,42]. This is followed by DNA end-processing, gap filling and ligation. Deficiencies in different factors involved in SBBR result in severe neurodegenerative phenotypes. Patients with defects in the DNA end-processing factors aprataxin, tyrosyl-DNA-phosphodiesterase 1 and polynucleotide kinase/phosphatase develop different types of cerebellar ataxia and microcephaly with sensitivity to genotoxic agents [43–50].

Oxidative and helix-distorting lesions are common types of DNA damage and are mostly repaired by three major excision repair pathways. In one of these pathways, base excision repair (BER), a DNA glycosylase recognizes and excises small chemical modifications such as oxidative lesions and small alkylation products and SSBs triggering a downstream repair signalling cascade [51,52]. BER is the main mechanism countering the deleterious effects caused by ROS, often regarded as drivers of the ageing process. It is possible that BER dysfunction plays a significant role in age-related phenotypes as it has been shown that several tissues in old mice display reduced BER capacity [53]. Importantly, age-associated neurodegenerative diseases such as Alzheimer's and Parkinson's diseases have also been linked to increased oxidative DNA damage [51,54,55] and BER has been shown to be impaired in the brains of sporadic Alzheimer's disease patients [56].

A second pathway, mismatch repair (MMR), corrects base mispairs and short deletion/insertion loops originated from replication errors, thus becoming a critical system ensuring maintenance of genome stability following DNA replication [57].

The last of the major excision repair pathways, nucleotide excision repair (NER), removes helix-distorting DNA lesions in four consecutive steps: (i) lesion recognition (ii) DNA unwinding (iii) damage excision and (iv) DNA synthesis and ligation. Two distinct lesion-recognition systems initiate the same downstream machinery, allowing the differentiation of two NER sub-pathways: (i) transcription-coupled NER (TC-NER), activated by RNA polymerase II stalling during transcription by chromatin remodelling proteins Cockayne's syndrome protein A (CSA) and B (CSB), and (ii) global genome NER (GG-NER), initiated by the UV-damaged DNA-binding protein (UV-DDB) complex and xeroderma pigmentosum group C (XPC) protein, which scan the entire genome, independently of transcription [58].

Syndromes caused by inherited defects in the NER machinery are rare and are surprisingly heterogeneous in terms of symptoms, despite a common feature of hypersensitivity to sunlight. Defects in the NER enzyme genes XPA, XPB, XPC, XPD, XPE, XPF and XPG can cause xeroderma pigmentosum owing to a defective GG-NER. GG-NER defects lead to the accumulation of lesions across the entire genome and, therefore, it is not surprising to observe that patients with xeroderma pigmentosum, in addition to sun-induced pigmentation abnormalities, also display a dramatically increased risk of skin cancer and internal tumours [59]. By contrast, defects in TC-NER do not inherently lead to an increased mutational load instead, cells remain in a state of blocked transcription that ultimately leads to apoptosis. Mutations in the aforementioned lesion-recognition genes CSA and CSB can cause Cockayne's syndrome (CS). Patients with CS display a range of symptoms associated with accelerated ageing, including growth/development impairment, severe neurological defects, hearing loss, cataracts and cachexia (for an extensive review of the clinical features, see [60]), reflecting the systemic consequences of the elimination of cells with low levels of transcription-blocking DNA lesions. In addition, specific point mutations in the NER helicase genes XPB and XPD can also cause trichothiodystrophy, a severe progeroid syndrome in which patients display the features of CS and also brittle hair and nails [59,61]. The features of these NER-deficiency syndromes are well studied in animal models [62–66]. Importantly, studies with animal models have revealed that the severity of the progeroid features correlates well with the degree of DNA repair defects, suggesting causality [66,67].

Lastly, highly cytotoxic DSBs are primarily repaired either by the efficient but error-prone nonhomologous end-joining (NHEJ) pathway or the more precise homologous recombination (HR) pathway. NHEJ works in somatic cells (or proliferating cells in G1 stage) and is capable of joining the ends of the DNA strand via different sub-pathways, depending on the configuration of the DNA ends [68] however, it works without a proper template, as it occurs independently of replication, and, therefore, often results in mutations (deletions or insertions). On the other hand, HR works in proliferating cells and is of particular importance during embryogenesis. After replication, HR uses the available identical copy of the damaged DNA to properly align the broken ends and repair the lesion [69], thus promoting cell survival without contributing to an increased mutagenic load. In addition, together with Fanconi's anaemia (FA) proteins, HR is also involved in ICL removal. In humans, mutations in key HR genes lead to a clear increase in cancer development, with mutations in the BRCA1 and BRCA2 genes being associated mostly, but not exclusively, with breast and ovarian cancer [70–72]. Mutations in HR genes can also lead to the development of FA/FA-related pathologies, characterized by bone marrow failure, developmental deficiencies and also an increased risk of cancer development [70].

The existence of such complex syndromes highlights the intricate relationship between DNA damage, ageing and cancer predisposition. Mutations in repair pathways that deal with mutagenic lesions often lead to cancer development while mutations in systems dealing with cytotoxic lesions (i.e. arrested transcription) are detrimental for normal growth and tissue homeostasis and thus contribute to an ‘artificially accelerated’ ageing process. This phenotypical dichotomy emphasizes the trade-off between the decisions a cell needs to take when facing irreparable DNA damage: minimize malignancy risk or maintain tissue functionality.

Moreover, the broad range of, sometimes highly specific, pathological outcomes strongly suggests that those genome instability syndromes, and physiological ageing itself, cannot be explained simply by the cell-autonomous effects of DNA damage. If the cell-autonomous DNA damage-induced increase in mutagenesis/cell death were the sole agent at play here, one would expect to observe similar tissue-unspecific pathologies independently of the affected repair pathway. Instead, different types of DNA damage appear to affect tissues differently, suggesting that complex signalling pathways might influence the whole organismal phenotype by coordinating specific systemic responses to damage.

5. Non-cell-autonomous DNA damage responses

The direct cell-autonomous consequences of DNA damage are unlikely to be the sole cause of both the complex pathological outcomes observed in patients with genome instability syndromes and the broad range of age-related phenotypes. Under this paradigm, the following questions arise: (i) Are there non-cell-autonomous responses promoting tissue dysfunction following DNA damage? (ii) Alternatively, are there compensatory non-cell-autonomous responses aiming to re-establish tissue homeostasis? (iii) Can different types of damage elicit specific systemic responses?

Regarding (i), we discuss below the influence of cellular senescence in the ageing process. Accumulating evidence shows the contribution of cellular senescence to age-related tissue dysfunction, and ablation of senescent cells via different mechanisms has shown potential in ameliorating multiple age-related phenotypes and even increasing lifespan [73–76]. Additionally, because of their heterogeneous aberrant secretory profiles (the senescent-associated secretory phenotype, SASP) [77,78], senescent cells are indeed capable of coordinating distinct non-cell-autonomous responses able to disrupt tissue homeostasis. Moreover, the tight links between cellular senescence, inflammation and stem cell exhaustion reflect the entanglement between different hallmarks of ageing and how multiple physiological layers orchestrate the onset of age-related functional decline.

Finally, regarding questions (ii) and (iii), we discuss recent findings in C. elegans and in mammalian models exemplifying compensatory stress responses, involving trans-tissue communication, elicited following different types of damage in order to maintain tissue functionality.

5.1. Cellular senescence, inflammation and ageing

Cellular senescence has been traditionally regarded as a state of irreversible cell-cycle arrest elicited by replicative exhaustion (replicative senescence) or in response to diverse, oncogenic or DNA-damaging stressors (oncogene-induced senescence and stress-induced premature senescence) [79–82]. Classical hallmarks of senescence include (i) promiscuous and highly heterogeneous gene expression (ii) apoptosis resistance and (iii) growth arrest [82]. For decades now, cells have been mainly identified as senescent by the presence of senescence-associated β-galactosidase (SA-βGal) activity [83]. Senescent cells display striking changes in gene expression when compared with their non-senescent counterparts [84–86] major changes often include the overexpression of important cell-cycle inhibitors, including the cyclin-dependent kinase inhibitors p21 (CDKN1a/CIP1) and p16 (CDKN2a/INK4) [82,87–89], downstream effector proteins resulting from the activation of p53, thus linking the DDR to the establishment of cell-cycle arrest. In recent years, many other features have been associated with a senescent phenotype, including loss/redistribution of lamin B1 [90], accumulation of lipofuscin [91,92], loss of nuclear HMGB1 [93], telomere-associated DNA damage foci [94], senescence-associated heterochromatin foci [95] and senescence-associated mitochondrial dysfunction [96], to name but a few. Most striking, though, is the distinct secretome profile of senescent cells, termed SASP [97,98]. Senescent cells secrete a host of pro-inflammatory cytokines, chemokines, growth factors and matrix-remodelling enzymes capable of altering a tissue's microenvironment via autocrine and paracrine signalling, ultimately contributing to age-related tissue dysfunction [98,99]. Importantly, senescent cells, via the SASP and/or ROS production, have been shown to be capable of maintaining a state of chronic inflammation and inducing senescence in adjacent bystander cells, both in vitro and in vivo [100–104]. This induced senescence, via a ‘bystander effect’, might be a relevant mechanism leading to the reported age-dependent accumulation of senescent cells in vivo [105,106]. Even more worrisome, by creating a local chronic state of inflammation via the SASP, a small amount of senescent cells can convert other, otherwise healthy, cells into a senescent state, which in turn will escalate the production of pro-inflammatory components, creating a positive feedback loop potentially capable of affecting the organism in a systemic way (figure 2). This is especially concerning, as it has been reported that even relatively low numbers of senescent cells induce a bystander effect and can disrupt tissue homeostasis [75,104,107,108] thus, senescent cells might play an active role as a driver of many age-related disorders and the process of physiological ageing itself.

Figure 2. Non-cell-autonomous DNA damage responses contributing to age-associated tissue dysfunction. Cellular senescence can be elicited in response to a permanent DDR following exposure to DNA-damaging agents (a). Once established, senescent cells secrete a host of pro-inflammatory cytokines, chemokines, growth factors and matrix-remodelling enzymes (SASP), capable of coordinating distinct non-cell-autonomous responses. Via the SASP, senescent cells create a local pro-inflammatory environment that can reinforce their own senescent state (autocrine senescence) and, simultaneously, induce senescence in bystander cells (paracrine senescence) (b). This induction of senescence in bystander cells might be a relevant mechanism contributing to the reported age-associated accumulation of senescent cells in multiple tissues. Additionally, the resulting pro-inflammatory environment (c) might create a positive feedback loop, escalating the number of senescent cells within a tissue and the production of pro-inflammatory components, contributing to age-associated tissue dysfunction.

The contribution of senescent cell accumulation to ageing is very well illustrated by the studies of Baker et al., originally with BubR1 mice. BubR1 mice have severe genome instability due to defects in spindle assembly and, consequently, display multiple progeroid features and increased levels of senescent cells [73,109]. Notably, genetic clearance of p16-positive cells in the BubR1 background delayed both the onset and progression of age-related phenotypes [73]. Using the same system, clearance of naturally occurring p16-positive cells in a non-progeroid background increased both healthspan and lifespan [74]. These studies provide strong evidence that senescent cells are active drivers of the ageing process nevertheless, it is important to note that senescent cells can be highly heterogeneous and not every senescent cell shows necessarily increased expression of p16, so it is possible that only a number of specific senescent cell populations (in this case, p16-expressing cells) are the actual drivers of the observed phenotypes.

Intriguingly, senescence-like phenotypes have been reported in postmitotic cells such as neurons [110], osteocytes [111], retinal cells [112], myofibres [104] and cardiomyocytes [113], among others, challenging the traditional view of senescence as a proliferation arrest-dependent programme.

Senescence is, however, not only tightly associated with physiological age-related phenotypes but also with multiple age-associated pathologies [114]. Cells with senescence-like phenotypes have been shown to accumulate in the lung in cases of idiopathic pulmonary fibrosis [115], in osteoarthritic joints [116] and in the liver in non-alcoholic fatty-liver disease [117], among other situations [114]. Correspondingly, transplantation of senescent cells can induce an osteoarthritic-like phenotype and impair function [75,116]. Conversely, removal of senescent cells with senolytic drug treatments has been shown to improve several healthspan parameters [75] and to improve tissue function in animal models of pulmonary fibrosis [115], atherosclerosis [118], hepatic steatosis [117] and obesity [119].

Senescence can be a highly heterogeneous phenotype, induced by multiple inputs and often leading to different and even opposite outputs [99]. In fact, senescent cells have been shown to promote both tumour suppression [80,120,121] and tumour progression [107,108,122,123], to contribute to wound repair [124] (but ultimately they may also drive the ageing process) and have been associated with numerous age-associated disorders [114]. Cellular senescence can thus be classified as an antagonistic hallmark of ageing [27] and a good representative of the antagonistic pleiotropy theory of ageing. Cellular senescence might have evolved as a tumour suppression mechanism with clearly beneficial effects early on however, as senescent cell frequencies increase with age [105,106], the deleterious effects, mainly of the SASP, start to outweigh the initial, ‘selected-through-evolution’, beneficial effects.

The effects of the SASP are also inherently interconnected with other age-associated features, particularly the age-dependent increase in low-grade systemic inflammation (inflammageing) [125,126]. This increase in inflammation is not exclusively due to the SASP enhanced activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription factor, the gradual inability of the immune system to remove sources of inflammation and autophagy dysfunction are also important contributors to age-associated inflammageing [127,128]. Overexpression of NF-κB is a known driving feature of ageing [129] and its inhibition delays cellular senescence and the onset of age-associated features [130]. Conversely, chronic activation of NF-κB induced by knockout of its nfkb1 subunit promotes cellular senescence by exacerbating telomere dysfunction and reduces the regenerative potential of tissues, thus accelerating ageing [100]. The reduction of tissues' regenerative potential is also one of the most striking features of ageing. This results mostly from functional exhaustion of stem cells in the majority of an organism's stem cell compartments [27], promoted not only by inflammation [100,131] but also by DNA damage [132] and cellular senescence [133,134], once again highlighting the entanglement of mechanisms and feedback loops driving the ageing process. Moreover, NF-κB activation, specifically in the hypothalamus, has been shown to inhibit the production of gonadotropin-releasing hormone, consequently contributing to the dysfunction of several other tissues [135]. Additionally, the age-associated decrease in hypothalamic stem cells and decrease in exosome secretion also accelerate the ageing speed and distal tissue dysfunction [135,136]. The hypothalamus thus appears to be involved in modulating systemic responses driving age-related pathology [137], reflecting the importance of the brain during ageing.

5.2. Compensatory stress responses, tissue functionality and longevity

Numerous studies from the past few decades have revealed that the ageing process is genetically regulated and cannot be explained simply as a consequence of damage accumulation. Pioneering studies with C. elegans have shown that lifespan can be significantly extended by mutations in single genes [138]. Currently, hundreds of genes have been identified in model organisms in which mutations lead to an increased lifespan—in some cases, up to a 10-fold increase [139]. Identification of these genes allowed the recognition of multiple so-called ‘longevity-pathways’, which, importantly, appear to be evolutionarily conserved in mammals. Among the best studied of these pathways are the above-mentioned CR and the insulin/insulin-like growth factor 1 (IGF-1) signalling (IIS) pathways.

The mechanisms accounting for the beneficial effects of CR remain somewhat elusive but their study is paramount in identifying cellular/systemic processes counteracting the age-associated increases in morbidity and mortality. Both the IIS and the target of rapamycin (TOR) pathways have been shown to mediate some of the beneficial effects of CR [23,140]. The inhibition of the TOR pathway, in particular, has been shown to have a very well-conserved role in mediating the CR-dependent lifespan extension among different organisms [141]. Reducing TOR activity leads, among other processes, to an increase in autophagy and a decrease in protein biosynthesis, both required for the CR-dependent increase in lifespan [23,141–143]. Nevertheless, the exact mechanisms by which these two processes exert their effects are still poorly understood.

Curiously, classical studies have shown that mice under CR are also more resistant to different acute stressors, including damage by surgical procedures, toxic drug administration and acute increase in ambient temperature [144], highlighting the intrinsic link between somatic maintenance and the retardation of the ageing process. In particular, studies in both humans and other animal models have shown that CR ameliorates oxidative damage, in particular to DNA and RNA [145,146], improves cellular quality control by promoting autophagy [147], promotes mitochondrial biogenesis [148] and impairs the SASP of senescent cells [149]. These and other mechanisms may very well be the drivers of the observed CR-associated increase in lifespan.

Regarding the IIS pathway, in mammals, the production of IGF-1 is promoted by the growth hormone (GH) produced and secreted from the pituitary gland. Dampening insulin signalling by manipulating components of this pathway (i.e. GH, the insulin/IGF-1 receptors or downstream effectors, such as FOXO) has been associated with an increase in longevity in both animal models and humans [150–152]. In C. elegans, dampening of the IIS pathway via mutations in the daf-2 and age-1 genes (the genes encoding the worm's orthologues of the insulin/IGF receptor and phosphatidylinositol 3-kinase, respectively) results in lifespan extension [138,139,150]. This effect is, however, dependent on the DAF-16 (the FOXO orthologue) transcription factor [138,150], the main IIS effector.

Importantly, the IIS pathway responds to DNA damage [153,154]. DAF-16 has been shown to be activated in response to persistent DNA damage in somatic tissues during the development of C. elegans in order to promote tissue functionality and allow growth to proceed however, DAF-16 responsiveness to DNA damage is severely blunted with ageing [153]. Similarly, a recent proteome analysis of NER-deficient worms following UV exposure identified DAF-2 as a central hub, connecting different signalling nodes and coordinating a systemic response to permanent DNA damage [154]. Notably, the observed proteome changes resembled the ones naturally occurring during ageing [155,156], again underscoring the role of DNA damage accumulation during physiological ageing and hinting towards a systemic adaptive ‘survival response’ aiming to maintain tissue functionality following damage. Additionally, multiple transcriptome analysis of NER-deficient mice also showed a dampening of the somatotropic axis in response to DNA damage [157–160], emphasizing that the same physiological mechanisms are shared between short- and long-lived models [159,161]. Of note, this same response was also observed in wild-type animals exposed to DNA-damaging agents [157]. These findings raise the intriguing possibility that low levels of, possibly localized, genotoxic stress are capable of triggering systemic stress responses and actually contribute to the maintenance of tissue functionality. Supporting this hypothesis, NER deficiency in C. elegans has been shown to increase the expression of ‘stress-responsive’ genes and further increase the lifespan of the already long-lived daf-2 mutants [162].

Systemic responses to tissue-specific DNA damage have previously been observed in both Drosophila melanogaster and C. elegans [163,164]. In D. melanogaster larvae, DNA damage in the epidermis triggers an immune response dependent on c-Jun N-terminal kinase (JNK) and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling, consequently limiting insulin-like peptide secretion by the central nervous system and activating FOXO [163]. JNK signalling is a prominent stress-responsive pathway in D. melanogaster and JAK/STAT signalling has been shown multiple times to be involved in systemic responses promoting tissue regeneration in injury models [165,166].

In C. elegans, the germline has been known for many years to be able to influence somatic maintenance, with germ cell-deficient worms being long-lived and stress-resistant [167,168]. In addition, somatic tissues are able to respond to damage in germ cells. GG-NER mutant worms, unable to remove DNA lesions in the germline, have surprisingly resistant somatic tissues. This stress response is mediated by an innate immune response in the germline triggered by the mitogen-activated protein kinase 1 (MPK-1, the extracellular signal-regulated kinases 1/2 (ERK1/2) MAPK homologue), which later becomes systemically established. This coordinated mechanism has been termed ‘germline DNA damage-induced stress resistance’ [164] and appears to be a mechanism evoked in order to extend an organism's lifespan following localized DNA damage. This provides the organism with more time to repair the damage present in the germline and prevent the transmission of harmful mutations to the next generation without sacrificing too much of the amount of progeny produced [169]. Supporting this explanation, offspring production is transiently reduced following exposure to DNA-damaging agents, but resumes after the period used for DNA repair and lasts past the normal reproductive period of non-damaged worms [164].

Lastly, it is important to mention the role of the neuronal system in the coordination of systemic stress responses. Neurons are ideally equipped for this as they are capable of (i) secreting chemicals able to reach distal tissues (ii) sensing and processing environmental cues and (iii) integrating those signals and coordinating physiological responses accordingly. Many reports have demonstrated the crucial role of the neuronal system in regulating longevity and proteostasis in C. elegans, mostly via communication with the intestine, presumably via neuroendocrine signals [170–178]. In the future, it would be of importance to better characterize the types of neuronal responses elicited following distinct DNA lesions and investigate possible neuron-mediated distal stress responses following DNA damage.

6. Concluding remarks

Research over past decades has elucidated the role of genomic instability as a root cause of ageing. The observed age-dependent accumulation of somatic mutations in the genome [26] and the accelerated ageing phenotypes caused by deficiencies in DNA repair systems provide compelling evidence supporting an active role for intrinsic DNA damage in mediating loss of tissue functionality with ageing. Still, the broad range of phenotypic variability within ageing populations strongly suggests that complex signalling pathways might coordinate specific systemic responses to DNA damage. These systemic responses have become increasingly apparent in multiple species and appear to have a major role not only during the physiological ageing process but also in response to acute stress. Importantly, these responses represent perfect examples of the intricate connection between DNA damage and other hallmarks of ageing, such as cellular senescence, stem cell exhaustion and altered intercellular communication [27], which can all occur as a consequence of the DDR. Nevertheless, the interplay between cell-autonomous and these non-cell-autonomous responses is still somewhat poorly understood. Future studies should aim to better understand how different types of DNA lesions can elicit such phenotypic variability by identifying key effector systemic signalling networks. For this, C. elegans might prove especially useful, as the nematode worm is an ideal model organism to study the consequences of damage in fully differentiated, postmitotic tissues. This system will facilitate the identification of mechanisms orchestrating the systemic consequences of DNA damage and will surely provide important novel insights about the impact of genome instability in physiological ageing and age-related pathology.

Sex differences in biological aging

While a growing body of evidence is accumulating on the relevance of biomarkers of aging in human health and mortality, understanding the sex-specific features of these markers is lagging behind. Not only has the effect of sex been largely ignored but is also often considered a confounder rather than a source of biological variation. Treating sex merely as a confounder or a ‘nuisance parameter’ can lead to results that are not biologically relevant to either sex. In the following sections, we discuss the available literature on sex differences in humans, with supportive evidence from animals, for the most commonly studied biological processes and markers of aging and highlight the key lessons learned from these studies so far. An overview of the topic and a conceptual framework is presented in Table 1 and Figure 1.

Sex specificity in human biological aging and associated theories.
Human biomarker of agingSex-specific effectsReferencesAging theoriesSexual dimorphism theories
Genetic factors in agingSex chromosomes,
X-chromosome inactivation in women,
Loss of Y in men,
Common genetic variants for anthropometric traits,
Transcriptional regulation
Bernabeu, 2020 Forsberg, 2017 Gentilini et al., 2012 Randall et al., 2013Senescence theory of aging:
1. Disposable soma
2. Mutation accumulation
Programmed theory of aging:
1. Developmental processes and growth
Sex chromosomes
Mitochondria-linked mechanismsBetter respiratory function in women,
Mutation accumulation,
Higher mtDNA abundance in women
Hägg et al., 2020 Demarest and McCarthy, 2015 Ventura-Clapier et al., 2017Senescence theory of aging:
1. ROS theory of aging
2. Mutation accumulation
Cellular senescenceMore senescent cells in male mice compared to females.Yousefzadeh et al., 2020Senescence theory of agingUnknown
Proteostasis and autophagyHigher proteasomal activity in female mice and fliesJenkins et al., 2020 Pomatto et al., 2017Senescence theory of aging:
1. ROS theory of aging
TelomeresLonger telomeres in girls/womenFactor-Litvak et al., 2016 Gardner et al., 2014Programmed theory of aging:
1. Hayflick limit
2. Developmental processes and growth
Senescence theory of aging:
1. ROS theory of aging
Sex chromosomes,
EpigeneticsHigher epigenetic age in boys/men,
Genome-wide DNA methylation and histone differences
Horvath et al., 2016 Horvath and Raj, 2018 Klein et al., 2019Programmed theory of aging:
1. Hayflick limit
2. Developmental processes and growth
Senescence theory of aging:
1. Disposable soma
2. Mutation accumulation
Sex chromosomes,
Inflammatory and immunological markersMen more affected by immunosenescence and inflammagingGubbels Bupp, 2015 Gomez et al., 2018 Franceschi, 2019Senescence theory of aging:
1. ROS theory of aging
Nutrient sensing and metabolismWomen have more beneficial (lower) fasting insulin levelsTempleman and Murphy, 2018 Pignatti et al., 2020 Comitato et al., 2015Senescence theory of aging:
1. The rate of living theory
Programmed theory of aging
Functional measuresMen perform better in physical functioning, regardless of the measuresPeiffer et al., 2010 Ganna and Ingelsson, 2015 Frederiksen et al., 2006 Finkel et al., 2019Senescence theory of aging:
1. ROS theory of aging
2. The rate of living theory
FrailtyWomen have higher levels, but men are more vulnerable to death at any given levelGordon et al., 2017 Gordon and Hubbard, 2019Senescence theory of aging:
1. Disposable soma
Leading causes of death (noncommunicable diseases) worldwide in 70 + year olds:
1. Ischemic heart disease
2. Stroke
3. Chronic obstructive pulmonary disease
4. Alzheimer's disease and other dementias
5. Diabetes mellitus
6. Trachea, bronchus, lung cancers
7. Kidney diseases
8. Hypertensive heart disease
9. Colon and rectum cancers
Men have higher incidence and death rates in:
1. Ischemic heart disease
5. Diabetes mellitus in midlife
6. Trachea, bronchus, and lung cancers
9. Colon and rectum cancers
Men have higher incidence of:
2. Stroke in early adulthood
5. Diabetes mellitus in midlife
Women have higher incidence and death rates in:
2. Stroke in late life
3. Chronic obstructive pulmonary disease
4. Alzheimer's disease and other dementias
7. Kidney diseases
8. Hypertensive heart disease
Women have higher incidence of:
5. Diabetes mellitus in youth
World Health Organization, 2021 Mauvais-Jarvis et al., 2020Programmed theory of aging
Senescence theory of aging
Sex chromosomes

Conceptual framework of the complex interactions between molecular, cellular, functional, organ, and whole body aging processes across the life course in men and women, with influences from chromosomes and hormones on the sex differences.

The different illustrations made for men and women are based on descriptions in the text. For healthspan and lifespan, trajectories are taken from a recent publication by Li et al., 2021.

Genetic factors in aging

The last two decades have been a revolution for human genetics, starting with the sequencing of a human genome in 2003 and breakthroughs in genome-wide association studies finding thousands of genetic loci associated with complex human traits, including many age-related diseases. For aging, gene discoveries have been sparse, although lately, large cohorts such as the UK Biobank have enabled powerful analyses of parental lifespan, healthspan, and longevity (Timmers et al., 2019 Zenin et al., 2019 Melzer et al., 2020). However, only a handful of genes have been identified, and the top loci are often well known for their relation to diseases, for example APOE, LPA, and CDKN2B-AS1. Longevity is known to be moderately heritable (Melzer et al., 2020) however, from an evolutionary perspective, natural selection is active for the reproduction of a species and not for maximizing lifespan. A recent study using human genotype data found that rare germline mutational burden was associated with lifespan and healthspan (Shindyapina et al., 2020). In particular, the association between mutations and healthspan was more pronounced in women. Another recent study found measured and genetically predicted levels of ten serum biomarkers to be associated with healthspan and lifespan, and again with stronger effects for healthspan seen in women (Li et al., 2021). Hence, many genes may be linked to the underlying aging process or beneficial for age-related diseases, with importance for longevity, health, and lifespan, but they may not have been specifically selected for (Rattan, 2000).

Thus far, large-scale genome-wide association studies have focused mostly on autosomes and rarely even stratified results by sex. Hence, little is known about sex-specific genetic effects for complex traits, although sexual dimorphisms have been reported for anthropometric traits and gout (Bernabeu, 2020 Randall et al., 2013), and gene-sex interactions have been found for multiple sclerosis (Traglia et al., 2017). Few efforts have been made for X chromosome-wide association studies, but they reveal (sex-specific) links to several complex traits and identify a locus associated with height escaping XCI (Bernabeu, 2020 Tukiainen et al., 2014). The mechanisms by which XCI is controlled are complex, for example by noncoding RNA and epigenetics (Lee, 2011), and are a way to balance the unequal amount of X-chromosomal DNA between men and women. The X chromosome encodes approximately a thousand genes, many related to metabolic activity, such as amino acid turnover and transport, and could explain the differential proliferative rates in sexes seen during embryonic growth (Patrat et al., 2020). During aging, the XCI ratio between maternal and paternal X chromosomes is no longer equal, leading to skewed XCI, which has been implicated in diseases and shown to be less severe in female centenarians (Gentilini et al., 2012). For men, the mosaic LOY in blood cells increases with age and is associated with age-related diseases and a higher risk of death (Forsberg, 2017).

Although the sex chromosomes are responsible for most of the female and male-specific traits, autosomes have bene increasingly studied for their role in sex-specific gene expression and associations with biological functions. Recent findings in this area point toward sexual dimorphism in transcriptomic profiles with hormone-related regulation and associations with various processes such as tissue morphogenesis, fat metabolism, cancer, and immune responses (Oliva et al., 2020). Implications on immunoinflammatory functions have also been highlighted (Bongen et al., 2019 Nevalainen et al., 2015). The underlying mechanisms for the sex differences in tissue specific transcription and associations with disease risks in men and women is currently unclear. However, there is some evidence that while most transcription factors have similar expression profiles in men and women, there may be sex-specific regulatory networks across different tissues, leading to altered function and disease control (Lopes-Ramos et al., 2020). For example, such sex-specific targeting patterns of transcription factors have been found for genes associated to Alzheimer’s disease (AD), Parkinson’s disease (PD), diabetes, autoimmune thyroid disease, and cardiomyopathy (Lopes-Ramos et al., 2020).

Genomic instability, such as chromosomal abnormalities, is known to be one of the hallmarks of biological aging (López-Otín et al., 2013). DNA damage accumulates across the life course as exogenous and endogenous triggers occur and DNA repair mechanisms become less efficient. Rare somatic mutations may accumulate across life and play a role in cancer, where men have been shown to have mutation accumulation earlier in life (Podolskiy et al., 2016), and in several premature-aging syndromes (Fischer and Riddle, 2018). Studies in rodents and in Drosophila support the association between DNA repair, mutational burden, and aging however, sex-specific effects are intricate, and the results depend heavily on animal strain and environmental conditions (Fischer and Riddle, 2018). Taken together, the examples described here relate to chromosomal stability and resemble well with the senescence theory of aging, where random events occur over time with less capacity of our maintenance system to repair and fix the faults. Sexual dimorphism in genome-wide studies for anthropometric traits may be consistent with the developmental processes and growth controlled during early life and aging, where hormonal influences are also apparent.

Mitochondria-linked mechanisms

Mitochondrial DNA (mtDNA) is inherited from mothers and contains the genetic code for 13 proteins, essential components of oxidative phosphorylation complexes, and several RNAs (Kauppila et al., 2017). Mitochondria are important for cellular processes such as energy production, oxidation, and apoptosis, and their function has been described as one of the hallmarks of aging (López-Otín et al., 2013). Mitochondrial dysfunction is associated with many age-related diseases (Ferrucci et al., 2020 Chocron et al., 1865). Oxidative damage and increased ROS production across life were initially thought to cause this dysfunction, but research in recent years showed that ROS do not accelerate aging in mice and even prolong lifespan in yeast and C. elegans (López-Otín et al., 2013). In humans, studies have linked the accumulated burden of mutations in mtDNA to aging and PD, although a majority of the mtDNA molecules within a cell must be affected for critical symptoms to emerge (Kauppila et al., 2017). Another feature of aging is the number of mtDNA copies within a cell. A lower number has been associated with aging, cognitive and physical decline, and increased mortality (Mengel-From et al., 2014). Historically, the free radical theory of aging, or the ROS theory of aging, has been postulated to explain mitochondrial dysfunction in aging (Gladyshev, 2014). However, evidence from both human and animal studies points toward the fact that the accumulation of mtDNA mutations is a feature of early life replication errors that undergo polyclonal expansion independent of ROS (López-Otín et al., 2013). The latter fits well with the whole senescence theory of aging (in which energy needs to be preserved to last across the full lifespan) and the mutation accumulation theory.

Substantial sexual dimorphism has been observed for mitochondrial function concerning oxidative capacity and enzyme activity (Ventura-Clapier et al., 2017). In humans, women show higher mitochondrial gene expression levels, protein content, and overall activity in multiple tissues, such as the brain, skeletal muscle, and cardiomyocytes (Ventura-Clapier et al., 2017). Similar sexual dimorphism is observed in rodent models investigating mitochondrial respiratory function (Ventura-Clapier et al., 2017). Estrogens have been shown to influence mitochondrial function and exert protective effects, partly explaining why women have delayed mitochondrial aging compared to men. These differences may contribute to altered mitochondrial function during stress conditions such as injury or starvation, where sex-specific effects are also noted on mitochondrial respiration (Demarest and McCarthy, 2015). Little is known about sexual dimorphisms in mtDNA copy numbers and accumulated mutations in relation to aging. A recent analysis in UK Biobank found that abundant mtDNA, estimated from the weighted intensities of probes mapped to the mitochondrial genome, was significantly elevated in premenopausal women compared to men and inversely associated with age, smoking, BMI, and frailty (Hägg et al., 2020). Hence, taken together, sex hormones likely play a pivotal role in explaining the beneficial effect seen in women on mitochondrial function and aging.


Telomeres are repeated sequences of nucleotide bases (TTAGGG)n located at the end of the chromosomes (Blackburn et al., 2015). Every time a cell divides, the DNA polymerase machinery replicates the DNA sequence into two identical copies, although the last part of the DNA is not preserved due to the end replication problem. Hence, instead of losing important coding materials, the telomere is shortened. When it becomes critically short, the cell enters senescence, and this was later found to be the explanation for the Hayflick limit (Olovnikov, 1996). However, germline cells have an active telomerase enzyme that elongates the telomeres to maintain length, as do many cancer cells, but somatic cells do not normally have this process. Therefore, throughout life, the length of the telomere (TL) decreases and serves as a marker of cellular aging (Blackburn et al., 2015). As different cells have varied rates of cellular turnover, the attrition rates of telomeres depend on the proliferative capacity of the host cell. Leukocyte TL is among the most proliferative cells with fast TL shortening, while skeletal muscle maintains longer telomeres (Demanelis et al., 2020). Increased attrition rates are seen in childhood and adolescence, when growth and development occur, as well as in old adults. In the elderly, cellular senescence is apparent where DNA maintenance and repair are no longer efficient, and telomeres reach critical lengths for cellular survival consistent with a person's natural lifespan limit (Steenstrup et al., 2017). As such, short TL has been associated with age-related outcomes and health aspects, for example mortality (Wang et al., 2018), CVD (Haycock et al., 2014), and different stressors in life (Starkweather et al., 2014). Telomeres are present across many species, but their length and attrition rates may vary (Oeseburg et al., 2010). Different genetic models have been used in mice to lengthen telomeres with telomerase activation, where some experiments increased the cancer incidence, while others did not (Folgueras et al., 2018). Recently, a model using hyperlong telomeres showed that this phenotype increases the lifespan in mice and shows overall beneficial effects on metabolism, glucose control, and mitochondrial function (Muñoz-Lorente et al., 2019).

The lengths of the telomeres are also sex-specific. At birth, boys have shorter TLs than girls (Factor-Litvak et al., 2016), which prevails throughout life (Gardner et al., 2014). As women have a longer lifespan than men, telomeres have been suggested as the causal factor explaining the difference. However, it is still not completely understood whether telomeres could be the cause or consequence of biological processes. Several large-scale genomic studies identified 30 + genetic variants associated with TL (Codd et al., 2013 Li et al., 2020a). These findings have led to increased knowledge, and many studies have provided evidence for causal associations between short leukocyte TLs and age-related diseases (Kuo et al., 2019). Hence, it seems that the biology of telomeres is a good example of how genes and the environment interplay to present a phenotype. Genetic liability contributes to the overall length of telomeres in all cells, and across the lifespan, stressors and lifestyle factors influence cell-specific attrition rates. Different aging theories may fit in this scenario, while the limit on cellular division (Hayflick) was described as a direct consequence of critically short telomeres.

The sexual dimorphism of telomere dynamics has been discussed in many different aspects (Barrett and Richardson, 2011). The sex chromosome-linked mechanisms could be part of the explanation. Although most telomere-related genes have been found in autosomal chromosomes, it has been suggested that the unguarded chromosome in heterogametic sex is a disadvantage for mortality and telomere maintenance. A mutation in the DKC1 gene on the X chromosome – a gene involved in telomere biology – is often seen in patients with dyskeratosis congenita, which leads to rapid TL shortening and reduced survival (Savage and Bertuch, 2010). Another explanation is that the larger sex has disadvantages in the cellular maintenance, oxidative stress reactions, and telomere function because cellular capacity is linked to growth. Consequently, men, who are generally taller than women, should suffer from worse telomere function. However, a recent meta-analysis investigated sex differences in TL across 51 vertebrate species and found no evidence supporting either the heterogametic sex disadvantage or the sexual selection hypotheses (Remot et al., 2020). The analyses, including TL dynamics in mammals, birds, reptiles, and fish, did not find associations to support sex differences in longevity. Hence, the true nature by which TL sexual dimorphism presents remains to be elucidated. The importance of sex hormones may need further scrutiny, as they influence the level of ROS production, which may interfere with telomere maintenance and elongation (Coluzzi et al., 2019). However, other theories have been discussed, and many factors are likely important for sex-specific telomere dynamics.

Cellular senescence

Another hallmark of aging is cellular senescence. The lifetime of a cell is limited, as described by Hayflick, and the fate of a cell depends on the type of cell and what signals it receives and the damage it is exposed to across life. Events such as critically short telomeres, oxidative stress, replicative errors, mitochondrial dysfunction, pathogen response, oncogene activation, and other stress sources may induce senescence of the cell with irreversible replicative arrest (López-Otín et al., 2013). This state causes a response of cytokines and other proinflammatory factors to be released, which may trigger downstream effects in the surrounding tissue and invoke a senescence-associated secretory phenotype (SASP) (Ferrucci et al., 2020). Cellular senescence is tightly linked with aging, has been well correlated with DNA damage, and an increasing number of cells are senescent in old tissues compared to young tissues in a study of liver tissue in mice (López-Otín et al., 2013 Khosla et al., 2020). However, it has been difficult to assess SASP in human studies since the phenotype markers are heterogeneous and not consistently available in the circulation. Nevertheless, the systemic accumulation of senescent cells in aging has been associated with many age-related diseases and conditions, such as frailty, both in humans and animal models (Ferrucci et al., 2020 Khosla et al., 2020 Schafer et al., 2020). Currently, there is also increasing evidence for the beneficial antiaging effect of senolytic drugs as potential treatments to remove senescent cells when abundant (Ferrucci et al., 2020). Hence, cellular senescence is a core mechanism in the senescence theory of aging, where cells and tissues accumulate damage across life but is also essential in the Hayflick limit's programmed theory of aging (Ferrucci et al., 2020 Khosla et al., 2020 Schafer et al., 2020).

No human studies specifically investigate the difference between men and women in cellular senescence, and evidence from other models is sparse. A recent study in mice suggested that male mice have a higher number of senescent cells across life compared to female mice (Yousefzadeh et al., 2020), although at the end of life, the proportion of female senescent cells is almost at the same level as in male mice. The notion of higher cellular senescence in males would be consistent with the shorter telomeres seen. Evidence points to the fact that female stem cells have an increased capacity for regeneration, self-renewal, and proliferation (Dulken and Brunet, 2015), in line with a more beneficial cellular aging route in females/women. The limited knowledge would nevertheless suggest that sexual dimorphism exists, where women maintain better cellular maintenance throughout the life course. Regardless, more studies on sex differential senescent mechanisms are urgently needed to learn about the biological aging processes therein.

Proteostasis and autophagy

Protein homeostasis, or proteostasis, is the body's ability to maintain control over protein synthesis, folding, stability, degradation, and removal through autophagy (Hipp et al., 2019). During aging, the balance in the protein machinery is lost and unfolded and misfolded proteins can aggregate and cause pathological conditions seen in diseases of (neuro)degeneration, AD, PD, and diabetes (Hipp et al., 2019). Oxidative stress and heat may increase conformational changes and induce cellular toxicity from accumulated protein aggregations. Under stressful conditions, the heat shock response is activated in the cell, and unbound chaperones are available to assist in stabilizing the protein network. A study by Ubaida-Mohien et al. found a decreased representation of chaperone proteins in old skeletal muscle tissue in healthy adults, although autophagy-related proteins were overrepresented (Ubaida-Mohien et al., 2019). Experiments in worms, flies, and mice have shown that overexpressing chaperones and heat-shock proteins are associated with an extended lifespan, whereas models deficient in parts of the chaperone-heat-shock system present accelerated aging phenotypes (López-Otín et al., 2013 Ferrucci et al., 2020). Moreover, autophagy becomes dysfunctional with aging. In model systems, abrogation of autophagy leads to neurodegeneration and shortens lifespan, whereas increased basal activity of autophagy increases lifespan (Leidal et al., 2018). In humans, long-lived families have a better-maintained autophagy system, and individuals under starvation exhibit enhanced autophagic flux (Leidal et al., 2018). Hence, declining proteostasis control in aging may be an effect of accumulated aggregates and dysfunctional autophagy, consistent with the senescent wear-and-tear theory of aging, including the ROS theory of aging.

A recent investigation analyzed proteasome activity across nine different tissues and found higher activity in female mice than in their male counterparts (Jenkins et al., 2020). The largest sexual dimorphism was observed in the small intestine and kidney, specifically in chymotrypsin-like proteasomal activity. In another study, female fruit flies were more tolerant to oxidative stress and showed increased proteasome expression and activity than male flies, although the resistance was lost with age (Pomatto et al., 2017). Overall, adaptations to maintain homeostasis seem to depend on both age and sex, although studies on the latter are still limited (Pomatto et al., 2018). Females studied in animals and model systems also exhibit more resistance to stressors, partly hypothesized to be due to estrogens' beneficial effects (Tower et al., 2020). Analyses on sexual dimorphism in human protein homeostasis and autophagy aging processes are still lacking, which is understandable, as efficient high-throughput methods are not yet available (Ferrucci et al., 2020 Pomatto et al., 2017).

Epigenetic alterations

The term ‘epigenetics’ means ‘on top of genetics’ and is a collective term for chemical modifications altering the activity of the gene transcription process without changing the DNA code itself. There are four major types of epigenetic mechanisms: ATP-dependent chromatin remodeling complexes, histone, and DNA modifications, and noncoding RNAs (Pagiatakis et al., 2021). Histones can be modified posttranslationally. The most well-studied mechanisms are acetylation and methylation processes changes in histone acetylation/methylation have been linked to aging, healthspan, and lifespan in diverse models, such as flies, mice, yeast, and human cell lines (Yi and Kim, 2020). A study by Klein et al., 2019 found that tau may affect histone acetylation in the human brain using an epigenome-wide association study of H3K9ac, thus relating histone modification processes to AD pathology. However, in human studies, genome-wide DNA methylation arrays have paved the way for a new field of research on epigenetic age where hundreds of (un)methylated sites (CpGs) have been shown to associate with age across the life course (Zhang et al., 2020). A multitude of clocks quantifying biological age across tissues, in whole blood, skin, muscle, or in human cell culture models have emerged (Horvath and Raj, 2018) and recently across mammalian species (Lu, 2021). With remarkable accuracy, clock ticks with aging and a higher epigenetic age are associated with worse health and increased mortality risk (Horvath and Raj, 2018 Chen et al., 2016). Promising studies have reported reversal of epigenetic age with different interventions (Horvath, 2020 Fahy et al., 2019). A still unanswered question is whether this reversal of the epigenetic clock would then infer a lower risk for adverse events. In other words, is the epigenetic process causal in aging (Zhang et al., 2020)? As with telomeres, the epigenetic clocks seem to be tightly linked with cellular replication underlying the Hayflick limit theory (Wagner, 2019). Genetic studies of epigenetic clocks have discovered several loci associated with lifespan and lifestyle factors beyond the gene regions where the CpGs themselves are located (Lu et al., 2018 McCartney, 2020). One of the top loci found harbors the telomerase TERT gene, demonstrating the link to telomere biology. Epigenetic changes have also been proposed due to both developmental and maintenance processes, where gestational age clocks represent the former and other adult tissue clocks represent the latter. Moreover, intrinsic and extrinsic epigenetic clocks have been suggested to represent internal (cellular) versus external (lifestyle stressor) aging processes (Horvath and Raj, 2018). The epigenetic process in aging may be consistent with both senescence and programming theories on aging depending on the specific timing in life and the clock under study.

The sex-specific effect on epigenetic age is apparent in young children and adults (Horvath et al., 2016 Horvath and Raj, 2018). At all ages, boys/men have a higher epigenetically predicted biological age than girls/women, in accordance with the survival benefit in women. This phenomenon seems to be true across different tissues and gives rise to an effective difference in mortality risk between men and women (Li et al., 2020b). Moreover, in women, earlier menopause, either natural or surgical, is associated with increased epigenetic age, and although the finding was not consistent across different tissues, there was further support for lower epigenetic age in women undergoing HRT (Levine et al., 2016). Little is known about sex-dimorphic effects on histone modifications in aging, although studies on different interventions and acetylation/methylation in animals suggest that these effects are important modifiers in aging (Fischer and Riddle, 2018). Furthermore, the Klein study found >4000 H3K9ac sites associated with sex in their human histone data, highlighting the future need for deeper studies in this area (Klein et al., 2019). Studies investigating genome-wide DNA methylation differences between men and women report significant differences in autosomes and on the X chromosome, the latter being linked to sexual dimorphism genes and XCI (Li et al., 2020c McCartney et al., 2019). A recent meta-analysis study investigating the age-related sex differences in DNA methylation patterns found changes associated with both methylation level and variability across the genome (Yusipov, 2020). Differentially methylated sites were enriched in imprinted genes but not in sex hormone-related genes. Furthermore, the top CpGs displayed a sex-specific pattern in samples from centenarians (healthy aging model) and Down's syndrome (accelerated aging model). On the other hand, another study investigating brain DNA methylation patterns found no support for sex-age interaction effects in neurodegeneration from human samples on AD and controls (Pellegrini, 2020). Studies on sexual dimorphism and DNA methylation are sparse in animal models, but some evidence for differences has been found in both rats and mice (Sampathkumar et al., 2020). Bacon et al., 2019 used a rat model resembling human neuroendocrine function and showed that DNA methylation regulates the onset of menopause. Taken together, the sexual dimorphism seen in epigenetic studies on aging is complex and seems to reflect sex chromosome-linked mechanisms and/or hormonal biological processes.

Inflammatory and immunological makers

Immunoinflammatory functions are at the heart of health in aging, and there is exhaustive literature available on the various changes that take place with age. At a cellular level, two distinct yet often parallel processes characterize immune aging: immunosenescence and inflammaging. The former refers to changes in the adaptive immune system, such as increased numbers of memory CD8 +T cells (resulting in a decreased CD4/CD8 cell ratio), loss of the key costimulatory molecule CD28 on the T cell surface and compromised clonal expansion and specific antibody production in the B cell compartment (Gubbels Bupp, 2015 Franceschi, 2019). Inflammaging refers to chronic, low-grade inflammation that occurs in the absence of infection and manifests as increased production of proinflammatory cytokines, linked to both frailty and CVD (Ferrucci and Fabbri, 2018). From an evolutionary perspective, inflammaging can result from positive selection of genetic variants that associate with higher levels of pro-inflammatory factors and enhanced immune responses in early life, conferring better protection against pathogens but resulting in increased damage to host tissues in later life. Inflammaging is thus in accordance with multiple different theories, where various stimuli, such as oxidative stress and lifestyle factors, contribute as well (Franceschi, 2019 De la Fuente and Miquel, 2009).

While both sexes experience aging-associated changes in the immune system, the hallmark features differ for men and women, and men are considered to experience maladaptive changes to a greater extent (Gubbels Bupp, 2015 Gomez et al., 2018). Between puberty and menopause – when differences in the hormonal milieu are the greatest between men and women – women experience lower rates of infections, an advantage attributed to stronger immune and vaccine responses and more efficient pathogen clearance (Gubbels Bupp, 2015). On the other hand, women are more susceptible to autoimmune diseases than men. However, after the age of menopause, the incidence of autoimmune diseases in women decreases close to the numbers observed in men, whereas the incidence of chronic inflammatory diseases increases (Gubbels Bupp, 2015). The temporal dynamics of these changes point to the crucial role of sex hormones in shaping immune aging, although it is likely much more complicated, involving an interplay of multiple homeostatic systems. It has been shown that nonimmune cells, such as adipocytes, fibroblasts, and endothelial cells, also contribute to inflammaging (Franceschi, 2019). As stated above, men seem to experience immunosenescence to a greater extent than women, potentially because women exhibit higher basal immunoglobulin levels, higher CD4 +T cell counts, and an increased CD4/CD8 T cell ratio compared to men (Gubbels Bupp, 2015 Gomez et al., 2018). The corresponding adaptive immune functions, such as antigen-specific antibody responses and CD4 +T cell cytokine production, are also typically more enhanced in women (Gubbels Bupp, 2015 Gomez et al., 2018). A recent study using sequencing and flow cytometry data in blood mononuclear cells further elucidated the sexual dimorphism in immune aging by showing that male and female cells also significantly differ at the age when sex hormones decline (Márquez et al., 2020). Older women had higher genomic activity for adaptive immune cells, while older men had higher activity for monocytes and inflammation, indicating greater inflammaging in men (Márquez et al., 2020). In the same study, a life-course analysis of the timing of epigenomic regulation of chromatin accessibility showed that male immune cells are more strongly affected and that a decline in immune function occurs 5–6 years earlier in men than in women (Márquez et al., 2020).

Although animal models cannot fully recapitulate human immunosenescence or inflammaging, findings on sex-related immune functions in animal studies have generally been in line with observations in humans. Sex differences are present in diverse species ranging from insects to mammals, with female individuals presenting stronger innate and adaptive immune responses than males (Klein and Flanagan, 2016). Like humans, the differences are largely attributable to the effects of sex hormones, with a contribution of genetic differences due to several immunoinflammatory genes that are X chromosome encoded (Klein and Flanagan, 2016). In summary, the above findings support the assertion that men experience faster and/or earlier aging-associated immunoinflammatory changes and that these changes may be attributed to both hormonal changes and other factors.

Nutrient sensing

Intracellular nutrient-sensing pathways and signaling systems mediate information on nutrient availability and energy levels in the extracellular milieu. The key pathways include the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway, mechanistic target of rapamycin (mTOR), and adenosine monophosphate-activated protein kinase (AMPK) pathway (Pignatti et al., 2020). These pathways regulate a multitude of intracellular functions, such as cell cycle control, DNA replication and repair, autophagy, and antioxidant defenses, by which their effects are excreted for reproduction, growth, and aging (Pignatti et al., 2020). Deregulated nutrient sensing is also one of the hallmarks of aging (López-Otín et al., 2013). Each of the hallmarks of aging is associated with undesirable metabolic alterations (López-Otín et al., 2016), stressing the fact that nutrient sensing and metabolism are interlinked processes with broad effects on whole-organism functions. Over the past years, there has been intensive research on how nutrient-sensing pathways control lifespan and healthspan, with the most significant breakthroughs achieved in unraveling how different dietary restrictions improve aging outcomes and survival in several species, including humans (Templeman and Murphy, 2018). Of the different dietary restrictions, the most compelling evidence rests on caloric restriction (CR), in which the energy intake is reduced

30% relative to ad libitum-fed animals without reducing the intake of micronutrients (Templeman and Murphy, 2018). At the molecular level, CR triggers activation of stress response pathways that in turn reduce inflammation and increase repair and antioxidative functions. Interestingly, genetic polymorphisms in genes encoding proteins in the insulin/IGF and mTOR pathways are among those that are robustly associated with longevity, such that variants associated with the lower basal activity of the pathways are associated with longevity (Pan and Finkel, 2017).

Sex hormones regulate several key functions in nutrient sensing and metabolism of glucose, amino acids, and proteins, and it is not surprising that men and women differ in several metabolic characteristics. At the molecular level, women have lower fasting insulin and glucose levels, lower basal fat oxidation, and higher fat use but lower consumption of carbohydrates during physical activity (Comitato et al., 2015). The most noticeable difference is the fat distribution at the phenotypic level, so that men tend to have more visceral fat, whereas women have greater fat deposition in lower body depots (Comitato et al., 2015). For healthspan, the above-described traits tend to favor women such that they have a lower risk of cardiometabolic diseases (before menopause). However, the higher basal insulin levels in men promote glycogen and lipid synthesis in muscle cells, resulting in higher muscle mass and strength (Comitato et al., 2015). Aging is, however, associated with a reduction in glucose tolerance in both sexes, increasing the risk of diabetes. There is a complex interplay between sex hormones and body composition for which data from in vivo studies and clinical trials remain inconclusive (Allan, 2014). Future studies will hopefully shed light on possible sex differences in CR in humans thus far, the available data do not support (or allow) inferences on sexual dimorphism. However, studies in rodents have suggested that males may have a more robust response to CR than females (Kane et al., 2018), but the mechanistic bases are not understood.

Akin to epigenetic clocks (see Epigenetic alterations) that predict mortality independent of other risk factors, there have been attempts to create similar composite measures based on metabolites measured using different techniques (Jylhävä et al., 2017). For example, Hertel et al., 2016 created a 'metabolic age score' that was shown to be associated with mortality independent of chronological age and other risk factors. The score was robustly associated with chronological age in both sexes, the only significant sex difference being that the score was more strongly influenced by obesity in women than in men (Hertel et al., 2016). However, such studies on metabolomics scores have been much fewer than studies on epigenetic clocks, and the potential sex dimorphism in metabolic scores is less clear. In summary, the sexual dimorphism in nutrient sensing and metabolism is largely attributable to sex hormones and their downstream effects. The higher muscle mass coupled with a higher basal metabolic rate in men also aligns with the rate of living theory.

Functional measures

Functional measures relevant to aging and mortality are numerous. One of the most commonly used and strongest markers for human population-based estimation of death risk is a simple assessment of walking speed (Ganna and Ingelsson, 2015), yet other popular measures include grip strength, chair rise, lung function, vision, and an abundance of cognitive domains (Peiffer et al., 2010). Although it is well known that being physically fit translates to better health, maintaining higher muscle mass and strength requires spending more energy and a higher metabolic rate. Analogous to the Hayflick limit, the rate of aging theory posits that the total amount of energy expenditure per lifetime is finite and that excessive usage results in accelerated aging (Pearl, 2011). Although much debated (Lints, 1989), this theory is supported by the observations that long-lived mammals have low energy expenditure rates, while short-lived mammals have higher rates. Studies in aging humans have shown that those having higher basic metabolic rates are more likely to die than those with lower rates (Ruggiero et al., 2008).

It is well established that men do better in physical capability, measured as grip strength, walking, and stair climb, even after adjusting for total body weight and lean body mass (Peiffer et al., 2010). Upon menopause, the withdrawal of sex hormones negatively affects bone and muscle health in women, where women experience a greater reduction in bone mineral density than men. However, men have a steady decline in bone function across life, but the interaction between load and bone strength is better maintained in older men, and this phenomenon may explain the reason for fewer fractures seen in men (Seeman, 2001). Women have less skeletal muscle mass than men, but men have greater loss with aging, although different parts of the body may show different sex-dimorphic effects, and menopause accelerates the loss in women (Doherty, 2003). Sarcopenia affects both sexes but is clinically more important in older women who may live longer with the disability (Doherty, 2003). For age-related visual impairment, women report more eye problems than men (Li et al., 2011), and overall, healthy adult men seem to perform better on visual perception than women (Shaqiri et al., 2018). In contrast, hearing loss is more frequent in men and may start as early as in the thirties (Shuster et al., 2019). Sexual dimorphism is also apparent in animal models, and women seem to be protected from age-related hearing decline before menopause, as estrogen levels are directly linked to the hearing threshold. Lung function is strongly associated with age, and a decline in spirometry-based measurements of dynamic flow starts soon after lung maturation in young adults (Sharma and Goodwin, 2006). Sex-specific differences are seen across almost all respiratory structures and functions women have smaller and anatomically different lungs than men, perform worse in breathing exercises, and sex hormones interact with lung and airway function during early developmental processes and aging (LoMauro and Aliverti, 2018). However, anatomical changes during aging to other organs may be advantageous to women. Cardiac remodeling due to aging is universal, but the decline in myocytes and systolic function are greater in males, both in humans and rodents (Keller and Howlett, 2016). Kidney function declines with aging, and men have a greater decrease in glomerular filtration rate, where women are most likely protected due to estrogens before menopause (Baylis, 2009).

A recent study created a composite measure, termed the functional aging index (FAI), to better capture the state and changes in various physical functions simultaneously. The FAI includes muscle strength (grip strength), movement (gait speed), sensory (vision and hearing), and lung function and is predictive of mortality in both sexes, yet the hazard ratio is greater in women (Finkel et al., 2019). However, while women had higher FAI scores than men, indicating poorer functioning, the rate of change did not differ between the sexes (Finkel et al., 2019). Hence, the better physical performance in men may be explained by evolutionary selection for physical fitness, which means better health in general, but it is unclear why this does not translate to a survival advantage. As men have higher muscle mass than women, some clues might be obtained from the observed associations between higher skeletal muscle mass and higher basal metabolic rate, that is energy expenditure that is higher in men than in women (Ruggiero et al., 2008). Perhaps, the sex specificity in functional measures best describes the complex interplay between fitness and aging in line with the rate of living theory in the senescence theory of aging, emphasizing the sex paradox in aging where women with worse physical function and health still outlive men, possibly due to a better cellular maintenance system and protections from estrogens.


Frailty is defined as a state of increased vulnerability to stressors resulting from decreased physiological reserves to maintain homeostasis across multiple organ systems. Manifestations of frailty overlap with those of normative aging yet are more pronounced. When a certain threshold in frailty is reached, the risk of adverse outcomes, such as disability and death, increases. Although frailty often coexists with multimorbidity (and disability), the association between frailty and mortality is independent of multimorbidity (Hanlon et al., 2018), indicating that frailty captures health-related variation that is not attributed to diseases alone. There is currently no widely accepted consensus on how to measure frailty however, the two most commonly used approaches are the Fried phenotypic model (FP) (Fried et al., 2001) and the Rockwood frailty index (FI) (Searle et al., 2008). The first views frailty as a physical syndrome with a discrete categorization of individuals into nonfrail, prefrail, and frail, whereas the latter considers frailty as a multidimensional construct based on the accumulation of deficits in physical, biological, and psychosocial domains. The FI is measured on a continuous scale, allowing for the detection of more subtle changes and making the FI suited for younger individuals. Although viewed more as a measure of fitness than biological age, frailty stands out as an exception in the wealth of research devoted to understanding the sex differences compared to the other markers. Women not only have a higher prevalence of frailty but also experience higher levels than men across the age range (Gordon et al., 2017). Women are nevertheless able to tolerate frailty better men are more vulnerable to death at any given level of frailty than women of the same age (Gordon et al., 2017 Jiang et al., 2017). The above-described male-female health-survival paradox may thus also be conceptualized as a sex-frailty paradox. The sex-frailty paradox has been described using several frailty scales (Theou et al., 2014) and across different populations (Gordon et al., 2017), suggesting that it is likely independent of the specific scale used to measure frailty.

The reasons for higher levels of frailty in women have been discussed previously, with various biological, social, and behavioral factors hypothesized to allow women to better tolerate frailty (Gordon and Hubbard, 2019 Hubbard, 2015). When conceptualizing frailty using the deficit accumulation model, that is the FI, it seems conceivable that women are evolutionarily ‘calibrated’ for late-life fitness. This theory aligns with the grandmother effect and increases in the population postreproductive lifespans when it benefits younger generations (Lahdenperä et al., 2004). Frailty also recapitulates characteristics of disposable soma theory that allow a certain amount of damage to the organism. However, another theory suggested underlying the sex differences is the chronic disease hypothesis by which women are more likely to experience nonlethal chronic conditions, while men tend to develop acute conditions associated with high mortality, such as stroke and myocardial infarction (Gladyshev, 2014 Bernabeu, 2020). Women may also be more prone to actively seek medical help for their conditions, resulting in better treatment balance of their (chronic) diseases. Last, variability in reporting behavior may contribute to the difference when using self-reported data, a common conception is that men tend to underreport their morbidities and disability, while women are more likely to overreport. However, evidence supporting this conception is not conclusive (Merrill et al., 1997 Macintyre et al., 1999), and the underlying mechanisms for the sex-frailty paradox remain unresolved.

In recent years, animal models of frailty, building on both FI and FP, have become available, providing opportunities to untangle how and why frailty develops and the mechanisms behind the sex differences. However, evidence on sex differences in frailty in animal models is less equivocal than in human studies. Few studies have reported that aged female mice exhibit higher FI scores than males (Heinze-Milne et al., 2019). However, other studies have reported no difference between the sexes, and one study found that male mice had higher FI scores than females (Heinze-Milne et al., 2019). The paucity of animal studies available and the variety in mouse strains used in the studies nevertheless warrant more evidence before the mechanisms of the sex differences in frailty can be resolved.

Sex differences in age-related diseases

Due to global aging and improved health care, the leading causes of death worldwide have shifted remarkably over the last century. Noncommunicable diseases, which are considered chronic age-related illnesses, are now the three most common causes of death worldwide (ischemic heart disease, stroke, and chronic obstructive pulmonary disease) (World Health Organization, 2021). For the population older than 70 years, all but one (lower respiratory infection) of the top 10 leading causes of death in the world are noncommunicable age-related diseases (Table 1 World Health Organization, 2021). An age-related disease can be defined as a disease where chronological age is a strong risk factor, and the incidence rate is increasing with increasing age. For a more comprehensive review on age-related diseases and the link to biological aging mechanisms, we refer to Franceschi et al., 2018. However, age-related diseases often present in a sex-specific manner. The top 10 leading causes of death by sex in those above 70 years reveals a change in the ranking of diseases so that instead of colon and rectum cancers, prostate cancer emerges in men and communicable diarrheal diseases in women. Hence, we highlight the sexual dimorphism in age-related diseases below, further strengthening the evidence that biological aging is different in men and women.

Although men and women present different disease-specific patterns and expression of risk factors, several leading age-related diseases are related to cardiovascular health in both sexes. It is well accepted that premenopausal women are relatively protected from the most common cardiometabolic manifestations, whereas postmenopausal women are not (Aggarwal et al., 2018). This observation has been attributed to estrogens' beneficial effects on CVD, metabolic syndrome, and diabetes. (For a more in-depth discussion on sex and gender aspects in aging diseases and treatment, we refer the reader to Mauvais-Jarvis et al., 2020 and Regitz-Zagrosek, 2012). In addition to looking at the sex hormones individually, several studies have shown that it may instead be the sex-specific testosterone/estradiol ratio that is more decisive on health outcomes than either of the hormones alone (Morselli et al., 2016). However, CVD is also tightly linked to inflammaging of the vasculature, and cellular senescence that could be reflected as TL shortening and intrinsic epigenetic age acceleration (Ferrucci and Fabbri, 2018). Hence, aging and sexual dimorphism in cardiovascular health are delicately intertwined.

Most cancers have apparent sex-differentiated effects, even after controlling for risk factors and lifestyle differences between sexes. In general, men have higher incidence rates and higher death rates in most cancers that are not related to reproduction (Mauvais-Jarvis et al., 2020). The male predominance is seen already in children with cancer before puberty, indicating that genetic or early developmental processes going wrong likely determine these differences. All cancer tumors have mutations in their genome, and commonly mutated genes are referred to as oncogenes (Stewart and Wild, 2014). There are many oncogenes known across the genome, some with specific X-linked mutational differences in men and women, and others encoded by the Y chromosome. Recent evidence suggests that noncoding genomic regions also contribute to sexual dimorphisms in driving cancer mutations and signatures (Li et al., 2020d). Many oncogenes present specific epigenetic signatures used for cancer diagnostics (Stewart and Wild, 2014), and epigenetic outlier burden is associated with age and cancer diagnosis in a sex-specific manner (Wang et al., 2019). Longer telomeres and extrinsic epigenetic age acceleration are also features seen in cancerous tissues. Hence, genomic instability, including the accumulation of mutations, epigenetic alterations, and telomere attrition, are hallmarks of aging and provide a link between aging and sexual dimorphism mechanisms in cancer. There are also cancers related to hormonal secretion where androgens are stimulating and estrogens are protective (Hammes and Levin, 2019). Cancers are not a class of homogenous diseases but complex, age- and sex-dependent biological processes that may arise due to several different factors.

AD and other dementias are perhaps the most established age-related diseases, and the prevalence continues to grow worldwide because of global aging. They are predominant in women, particularly in the oldest old, which may also be attributed to the female survival benefit (Mauvais-Jarvis et al., 2020 Mazure and Swendsen, 2016). There is evidence for sex-specific brain differences in early growth and development of the brain and adult structure and function, which may be of relevance to neurodegeneration. Cognitive aging in healthy adults demonstrates sex-differential effects, where men generally perform better in visuospatial ability and women better in verbal ability, but the speed of decline may be worse in men, although the literature is not consistent (Li et al., 2020b McCarrey et al., 2016). In AD, women present worse clinical symptoms for comparable levels of brain atrophy in men, and interactions with hormones may be one explanation for the differences (Toro et al., 2019). Early natural or surgical menopause and late initiation of HRT is associated with increased risk of AD (Mauvais-Jarvis et al., 2020). However, sex-differential effects may also be related to sex chromosomes. A recent study using an AD model in mice, expressing the human amyloid precursor protein, showed that adding an extra X chromosome decreased mortality and clinical AD symptoms (Davis, 2020). It should also be noted that sex differences in dementia incidence may be partially explained by selective survival (Shaw et al., 2021). Sex differences in the age-related diseases, frailty and domains of physical functioning are summarized in Figure 2.

Overview of the most significant sex differences in age-related diseases, functioning and frailty.

Abbreviations: AD, Alzheimer’s disease COPD, chronic obstructive pulmonary disease.

Thus, all the above calls for more research to better understand how biological sex and its attributes shape health in aging. Moreover, as many age-related diseases, most prominently CVD, are associated with systemic manifestations, such as low-grade inflammation, there is likely a complex bidirectional interplay between the diseases and biological aging at the cellular level. Having longer telomeres, for example, is protective for CVD and AD but a risk factor for many cancers, likely explained by the fact that tumor cells have overcome the problem of telomere shortening by activating the telomerase enzyme (Jylhävä et al., 2017). Epigenetic age has been associated with both cardiovascular and cancer deaths, depending on whether the clock represents intrinsic or extrinsic biological aging (Jylhävä et al., 2017). Hence, there is a trade-off between biological mechanisms promoting longevity and good cardiovascular health versus those promoting cancer growth. Therefore, more interesting than looking at the diseases or biological markers in isolation would be to assess the temporal dynamics between disease progression and aging biomarkers, with rigorous sex-specific approaches included.

Conclusions and Perspectives

A global view at the nine candidate hallmarks of aging enumerated in this review allows grouping them into three categories: primary hallmarks, antagonistic hallmarks, and integrative hallmarks ( Figure 6 ). The common characteristic of the primary hallmarks is the fact that they are all unequivocally negative. This is the case of DNA damage, including chromosomal aneuploidies, mitochondrial DNA mutations and telomere loss, epigenetic drift, and defective proteostasis. In contrast to the primary hallmarks, antagonistic hallmarks have opposite effects depending on their intensity. At low levels, they mediate beneficial effects, but at high levels, they become deleterious. This is the case for senescence, which protects the organism from cancer, but in excess can promote aging similarly, reactive oxygen species (ROS) mediate cell signaling and survival, but at chronic high levels can produce cellular damage likewise, an optimal nutrient-sensing and anabolism is obviously important for survival but in excess and during time can become pathological. These hallmarks can be viewed as designed for protecting the organism from damage or from nutrient scarcity, but when exacerbated or chronic, subvert their purpose and generate further damage. A third category comprises the integrative hallmarks, stem cell exhaustion and altered intercellular communication, which directly affect tissue homeostasis and function. Notwithstanding the interconnectedness between all hallmarks, we propose some degree of hierarchical relation between them ( Figure 6 ). The primary hallmarks could be the initiating triggers whose damaging events progressively accumulate with time. The antagonistic hallmarks, being in principle beneficial, become progressively negative in a process that is partly promoted or accelerated by the primary hallmarks. Finally, the integrative hallmarks arise when the accumulated damage caused by the primary and antagonistic hallmarks cannot be compensated by tissue homeostatic mechanisms. Because the hallmarks co-occur during aging and are interconnected, understanding their exact causal network is an exciting challenge for future work.

The proposed nine hallmarks of aging are grouped into three categories. In the top, those hallmarks considered to be the primary causes of cellular damage. In the middle, those considered to be part of compensatory or antagonistic responses to the damage. These responses initially mitigate the damage, but eventually, if chronic or exacerbated, they become deleterious themselves. In the bottom, there are integrative hallmarks that are the end result of the previous two groups of hallmarks and are ultimately responsible for the functional decline associated with aging.

The definition of hallmarks of aging may contribute to build a framework for future studies on the molecular mechanisms of aging as well as for designing interventions to improve human healthspan ( Figure 7 ). However, there are still numerous challenges ahead in relation to understanding this complex biological process (Martin, 2011 Miller, 2012). The rapid development of next-generation sequencing technologies may have a special impact on aging research by facilitating the evaluation of the genetic and epigenetic changes specifically accumulated by individual cells in an aging organism (de Magalhaes et al., 2010 Gundry and Vijg, 2012). These techniques are already being used to determine the whole-genome sequence of individuals with exceptional longevity, to perform comparative genomic studies between short-lived and long-lived animal species and strains, and to analyze age-associated epigenetic changes at maximum resolution (Heyn et al., 2012 Kim et al., 2011 Sebastiani et al., 2011). Parallel in vivo studies with gain- or loss-of-function animal models will be necessary for moving beyond correlative analyses and providing causal evidence in favor of the implication of these proposed hallmarks in the aging process. Besides the characterization of individual hallmarks, systems biology approaches will be required to understand the mechanistic links among the processes that accompany and lead to aging (Gems and Partridge, 2013 Kirkwood, 2008). Additionally, molecular analysis of the genome-environment interactions that modulate aging will help to identify drug targets for longevity promotion (de Magalhaes et al., 2012). We surmise that ever more sophisticated approaches for disentangling the complexities of normal, accelerated and delayed aging will eventually resolve many of the pending issues. Hopefully, these combined approaches will allow a detailed understanding of the mechanisms underlying the hallmarks of aging and will facilitate future interventions for improving human healthspan and longevity.

The nine hallmarks of aging are shown together with those therapeutic strategies for which there are proof of principle in mice.

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