The Role of Telomeres in Senescence, Aging and Disease: Fiction and Reality
Gabriele Saretzki *
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Newcastle University, Biosciences Institute, Campus for Ageing and Vitality, Newcastle upon Tyne, NE4 5PL, UK
* Correspondence: Gabriele Saretzki
Academic Editor: Muthuswamy Balasubramanyam
Special Issue: Perspectives on Telomeres and Aging II
Received: May 21, 2025 | Accepted: September 02, 2025 | Published: September 09, 2025
OBM Geriatrics 2025, Volume 9, Issue 3, doi:10.21926/obm.geriatr.2503324
Recommended citation: Saretzki G. The Role of Telomeres in Senescence, Aging and Disease: Fiction and Reality. OBM Geriatrics 2025; 9(3): 324; doi:10.21926/obm.geriatr.2503324.
© 2025 by the authors. This is an open access article distributed under the conditions of the Creative Commons by Attribution License, which permits unrestricted use, distribution, and reproduction in any medium or format, provided the original work is correctly cited.
Abstract
Telomeres are repetitive structures at the ends of linear chromosomes. Due to incomplete DNA replication at the end of linear DNA molecules, the so called “end replication problem”, telomeres shorten consecutively during cell division. In addition, telomere sequences are highly susceptible to oxidative stress damaging telomeres and resulting in their dysfunction even in non-dividing cells. Telomere shortening has been identified as one of the underlying causes for replicative senescence that can also contribute to aging due to the accumulation of senescent cells with advanced age in various tissues. These cells, in addition to an irreversible cell cycle arrest, are also characterized by a specific senescence-associated secretory phenotype (SASP) and hence increased inflammation. However, also postmitotic cells that do not shorten telomeres can have damaged and dysfunctional telomeres, undergo senescence without a specific cycle arrest and have a SASP. The influence of senescence on the aging process can be partially reversed by removing senescent cells from the body using senolytic agents. Many studies related to this topic, analyze predominantly blood leukocyte telomere length and suggest a direct causal relationship between telomeres, aging and diseases. Importantly, it is now more and more accepted that telomere length has a strong genetic element that is already obvious in newborns and might determine the trajectory of telomere dynamics through the entire adult life and thus pre-determine lifespan, aging and the susceptibility to various ageing-associated diseases. Moreover, there are additional factors such as oxidative stress and inflammation as well as lifestyle interventions that are able to influence telomere length (TL) and telomere shortening rate (TSR) during an individual’s lifetime. The review aims to raise awareness of the different factors that impact telomeres for a better understanding of the intricate relationship between telomeres, senescence, aging and age-related diseases.
Graphical abstract
Keywords
Telomeres; senescence; aging; DNA damage; oxidative stress; inflammation; age-related diseases
1. Introduction
Telomeres have become very popular during the last decades due to their presumed function as a biomarker of cellular senescence as well as organismal aging. However, there is a lot of confusion around in the scientific and in particular the clinical community, about the feature of telomeres as a marker for the health status, aging process and ageing-related diseases because telomere shortening (TS) has been described as an important hallmark of senescence and aging [1]. In various cross-sectional studies a correlation has been shown using blood telomere length (TL) as a predictive biomarker for mortality, morbidity and longevity in the human population [2,3,4,5]. Thus, this review aims to clarify a few misconceptions in this important field of health and disease. This includes the separation of the different parameters: telomere length (TL), telomere shortening rate (TSR) and telomere damage as well as telomere dysfunction. For the determination of those parameters the choice of the method has an important influence [6,7,8]. For example, TL can be determined by using a Southern blot-based method that measures the telomere restriction fragments (TRF) [9] or a qPCR method [10] that both mainly measure average TL and do not discriminate between different chromosomes within a cell and also not between telomere lengths in different cell types, for example, of a complex tissue or organ. Because TRF analysis includes using quite large amounts of DNA, restricting them with specific enzymes and running a Southern blot with additional detection methods such as DNA hybridization, this technique is only rarely used for large epidemiological studies. The vast majority of studies measures TL from white blood cells (PBMC, lymphocytes) using a relatively simple qPCR-based method due to its convenience as a high-throughput method when analyzing large sample numbers and because only small amounts of DNA are required [11]. Importantly, regarding the reliability and robustness of these two methods, it turns out that the more laborious southern blot TRF measurement method seems to be better for the quality and reproducibility of data than the more frequently used qPCR technique due to high variability of the latter method (>2%) both within and between samples [9]. However, both techniques only determine the average TL while the TRF analysis gives also an idea about the spread and heterogeneity of TL. Moreover, also sample sources and the DNA extraction method can have an important influence on the accuracy of TL measurement results [8,12].
A better discrimination of different telomere-related parameters can be achieved by using in situ techniques such as fluorescence in situ hybridization (FISH) techniques which are, except for FLOW-FISH, able to discriminate between different chromosomes [13,14]. Distinct techniques exist such as FISH on metaphases for in vitro cultures where each chromosome can be detected and the length of their telomeres determined [15]. Another FISH method is that on interphase nuclei which is predominantly used for tissues where also different cell types can be analyzed when using an additional histological marker for various cell types while a disadvantage is that not all chromosomes can be detected and thus also rather an average telomere length will be determined. A method mainly applicable for the analysis of blood cell TL is Flow-FISH where a flow cytometer is used and different cell types can be labelled in addition to telomeres [16,17]. However, all these FISH methods are rather laborious and only rarely, with the exception of Flow-FISH, used in a high-throughput format [18]. Importantly, individual telomeres on different chromosomes can have very different lengths as well as shortening rates [19,20] and senescence can be induced when one or several telomeres reach a critically short length and thus induce a DNA damage response (DDR) which can result in cellular senescence or apoptosis [21,22]. Moreover, it has been demonstrated that the frequency of short telomeres predicts mammalian longevity [23]. Thus, techniques such as TeSLA (Telomere Shortest Length Assay) which can be used together with TRF analysis, receive an increased interest in the field of telomere research as well as for their application in clinical diagnosis. In addition to average TL, TeSLA also determines the distribution and frequency of the shortest telomeres [24].
Interestingly, it is this group of the very shortest telomeres which apparently also gets predominantly but not exclusively, elongated by the enzyme telomerase [25] which is able to counteract telomere shortening in cells where it is active, while the longest telomeres seem to have the highest TSRs [26].
Importantly, as mentioned above, not just telomere length is an important read-out for the property of telomeres to signal a cell cycle arrest and senescence, but also the occurrence as well as the amount of unrepaired telomeric DNA damage which can accumulate anywhere in telomeres which form an important sentinel or damage sensor [27,28]. While telomere shortening is a mechanism that occurs only in dividing cells and is related to replicative senescence, telomeric damage can also form and accumulate in postmitotic cells and lead to a senescent phenotype that is not associated with an induced cell cycle arrest but still has many other features of cellular senescence such as heterochromatic foci [29,30], senescence-associated secretory phenotype (SASP) [31,32] etc. These features also arise in other senescence types such as stress-induced premature senescence (SIPS) which occurs under acute, mainly external stress [33] without changing TL, but most likely involving telomeric as well as general genomic DNA damage [34]. This form of telomere damage can be detected by using a DNA damage marker such as γH2A.X or 53BP1 which form DNA damage foci in addition to using telomeric FISH to identify the telomeres. Two major types of telomeric DNA damage can be discriminated from each other. The first type was initially described by T. de Lange as telomere-dysfunction dependent foci (TIFs) which mainly arises from telomeres getting very short and losing their capping function [35]. The molecular background for these TIFs will be detailed below. The second type of DNA damage predominantly arises within telomeres which can be long or short and is called telomere-associated foci (TAFs) [28,36,37]. Some scientists even argue that the longer a telomere is, the more DNA damage can occur [36]. This telomeric damage can also appear in postmitotic (PM) cells and result in senescence.
Another frequent misconception regarding telomeres is not to recognize the two main causes for their shortening and dysfunction. The first and most important is the already mentioned ERP which occurs during semiconservative DNA replication in dividing cells resulting in a consecutive shortening of telomeres (see details below). When only focusing on this mechanism, telomeres have been initially described as a “mitotic clock” which was historically used to describe replicative senescence but is rather outdated now while still cited in many studies and reviews today. However, pioneered by the work of T. von Zglinicki, the shortening rate of telomeres can be influenced by the level of oxidative stress [27,38]. When it is high, telomere shortening (TS) is accelerated up to 4-6 fold, while in the reverse case, it is less pronounced under decreased oxidative stress [2,39]. This stress can be external, due to toxins, radiation etc. or internal, for example due to inflammation processes within a cell, tissue or organism which can lead to higher generation of reactive oxygen species (ROS), predominantly from mitochondria, but also from other sources such as Noxes (NADPH-oxidases), which is then translated into higher DNA damage. Consequently, not just cell proliferation as a read-out for the number of mitotic cell divisions, but also the modification of the telomere shortening rate by oxidative stress should be considered when considering telomeres as a biomarker for senescence, aging or even ageing-related diseases. This is important to bear in mind and not considering these two factors for telomere shortening, often leads to contradictive study results or their incorrect interpretation. Moreover, the high sensitivity of telomeres to oxidative stress together with a low DNA repair capacity at telomeres are underlying mechanisms for persistent, unrepaired telomeric damage such as TAFs. Finally, various blood cells are able to activate telomerase activity (TA) thereby counteracting TS which might also be involved in complicating the interpretation of results from this cell type. This review aims to emphasize several frequently neglected features associated with telomeres, their length, shortening, damage and dysfunction and their relationship with senescence, aging as well as various age-related diseases.
2. Structure and Function of Telomeres
Telomeres consist of a DNA part- tandemly repeated hexanucleotide repeats ((TTAGGG)n in mammals) that is in humans around 5-15 kb long as shown in Figure 1 and a protein part, the shelterin complex with 6 proteins. Those bind either the double-stranded DNA (TRF1, TRF2) or the single-stranded 3’G-rich overhang (Pot1/ TPP1 heterodimer), as well as two other proteins (TIN2, Rap1) which bind to the other proteins and partly link them together [40,41,42]. The shelterin complex protects telomeres from degradation by nucleases and prevents the activation of the DNA damage response (DDR) [43]. The POT1/TPP1 heterodimer binds to the telomeric 3’ overhang that is formed during the telomere replication process and thereby suppresses the DDR [44]. The shelterin complex is a dynamic feature that can differ between cells and at different cell cycle stages [45]. Mutation or dysfunction of shelterin components results in the activation of the DDR, including phosphorylation of the histone H2A.X and ataxia telangiectasia mutated (ATM) [46]. In addition, shelterin is also instrumental for regulating the access of telomerase to telomeres and thus telomere length [47,48]. Shelterin also contributes to the determination of the specific set-point for maximal TL, thereby determining telomere homeostasis [49].
Figure 1 Basic structure of telomeric DNA at the end of linear eukaryotic chromosomes. Also indicated are the synthesis of TERRA-molecules from sub-telomeric promoters as well as the position of telomere-dysfunction-induced foci (TIFs) due to the exposure of the single-stranded (ss) G-rich 3’ overhang as well as telomere-associated foci (TAFs) anywhere at the length of a telomere.
The shelterin subunits TRF1 and TRF2 bind sequence-specifically to double-stranded telomeric DNA and are also involved in binding and recruiting additional shelterin components as well as non-shelterin proteins responsible for the maintenance of telomeric structure and function. Both TRF1 and TRF2 are involved in multiple functions at telomeres such as telomere protection, replication and telomere length homeostasis [50].
Due to the high amounts of guanines, these ends also tend to form G-quadruplex (G4) structures [51], which have to be resolved during replication in order to permit fork progression and to complete replication.
Importantly, telomeres form higher-order structures: D-loops and T-loops which contribute to the capping function of telomeres and prevent the activation of ATM and the DDR [52]. The D-loop (displacement loop) is formed from double-stranded telomeric DNA which tucks in the single-stranded 3’overhang with the help of Pot1 and TPP1 while the T-loop (telomeric loop) is formed by the telomeric double strand with the help of TRF2 and TRF1 ending as a lariat structure in the D-loop [44,53,54,55,56] (see Figure 2).
Figure 2 Loop structure and shelterin binding the telomere. The displacement loop (D-loop) is generated by tucking the single-stranded overhang into the telomeric double-strand. The telomeric loop (T-loop) is formed by the double-stranded telomere sequences. Shelterin proteins TRF1 and TRF2 bind telomeric double-stranded DNA and TIN2 binds to these two proteins. Rap1 binds to TRF2 and TIN2 also mediates the binding of TPP1 to Pot1 in order to protect the 3’ overhang.
The 3’overhang has a variable size of a few hundred nucleotides [57,58,59] and gets bound by Pot1 and TPP1 in order to protect it from degradation and from being recognized by the DNA repair system as a single-stranded DNA break [60]. In addition, this 3’overhang forms the substrate for the specialized reverse transcriptase telomerase which is able to add de novo telomeric repeats in late S-phase and thereby, in cells where it is active, to counteract and compensate for the telomere shortening from the end-replication-problem (ERP) [61].
The most important function of telomeres which are mainly heterochromatic, is to protect the coding genes of the chromosomes from degradation and thereby preventing chromosomal fusions that would eventually lead to genomic instability. Telomeres and coding regions are also further separated by subtelomeric regions which consist of some telomeric sequences interspersed with unique for each chromosome sequences (see Figure 1). These subtelomeres contain CpG islands and promoters which are able to synthesize long non-coding RNAs: TERRA (telomeric repeat-containing RNA) which are involved in the regulation of various telomeric functions, DNA repair as well as access of telomerase to telomeric ends [62]. TERRA hybridize with RNA forming hybrids called R-loops that intercalate into telomeric sequences and also promote the formation of G-quadruplex structures at telomeres [63]. While TERRA have various homeostatic functions for telomere maintenance and telomerase recruitment [64,65], they can be upregulated upon telomeric damage [66], are sensitive to oxidative stress and external factors from the environment [67,68] and seem to play a role in cancer cells [69,70]. TERRA are transcribed from many telomeres, with increased levels from short and dysfunctional ones [65]. Interestingly, TERRA, in particular those from chromosome 20q, have shown to be instrumental for telomeric epigenetics and heterochromatin assembly [71].
Moreover, telomeres can form even larger loops which via a so-called “telomere position effect” (TPE) influence the expression of chromosomal genes, even over long distances [72]. One could speculate that such TPEs might contribute to age-related changes in gene expression.
3. Causes for Telomere Shortening in Mitotic Cells
In dividing cells telomeres shorten during regular DNA replication due to the so called “end replication problem” (ERP) [73,74,75]. The ERP is based on the fact that DNA-polymerases are only able to continuously synthesize the leading strand in a 5’-3’ direction. Leading strand replication generates blunt ends that require 5′- to-3′ end resection by the exonuclease Apollo immediately after replication to generate an overhang. Thus, telomeres also undergo a nucleolytic end processing that can contribute to telomere shortening [76]. In contrast, the lagging, C-rich strand is replicated with the help of an RNA primer and short Okazaki fragments. The latter are eventually ligated together to form the newly synthesized lagging strand. However, at the very end of this new strand the most distal RNA primer is degraded whereby a gap is formed in the newly synthesised 5’C-rich DNA strand leading to a 3’G-rich single-stranded overhang at the old strand (see Figure 3). These processes result in the consecutive loss of telomeric DNA at each round of replication of around 20-100 bp in human fibroblasts in vitro under normal culture conditions, also depending on the individual antioxidant capacity (AOC) of the cell strain, presumably reflecting that of the donor organism [77]. This shortening rate can even increase up to 600 bp per population doubling (PD) of the culture under increased oxidative stress [2], while lowering oxidative stress can decrease the TS rate [78]. However, in vivo these values are less in leukocyte telomere length (LTL) with an average annual shortening rate of 30-35 bp [79] reaching an average total value of 5-6 kb TL at an age over 60 years [80].
Figure 3 Replication fork at the telomere. Shown are the continuous leading strand synthesis and the DNA synthesis of Okazaki-fragments with the help of an RNA primer which are then stitched together by a DNA ligase at the lagging strand.
An influence of the number of cell divisions onto the rate of TS has been detected when comparing the TSR between young children and adults. Benetos and colleagues compared the TSR in children below the age of 14 years and compared it to that of their parents. The study found that children shortened their LTL twice as fast as their parents from around 40 bp per year to around 20 bp/year with a high statistical significance [81]. Similar results were demonstrated in earlier measurements of unrelated individuals. Chen and co-workers have determined that lymphocytes lose around 30 bp of TL during adult life [82] while this shortening rate is significantly higher during the first 2 years of human life due to higher HSC replication rates [83,84]. Others have found that the specific telomere shortening rate, rather than the absolute telomere length, is a powerful predictor of species life span [85].
In addition, oxidative stress, DNA damage and modification of components of the shelterin complex can contribute to the modulation of telomere length (TL). Regulation of telomere length and their capping are essential for genomic stability [86]. In this process the shelterin complex plays an important role. For example, the Pot1/TPP1 heterodimer regulates the access of telomerase to telomeres in order for the former to extend and elongate telomeres de novo and possibly also to regulate the extent or the frequency of elongation [87]. Moreover, proteins that bind along the length of the telomere such as TRF1/TIN2/tankyrase form part of a negative feedback loop that regulates telomere length [44,47,88].
Oxidative stress, mainly in the form of reactive oxygen species (ROS) from mitochondria [89] inflicts DNA damage in the form of strand breaks [90] or oxidative modifications with 8oxoG being the most common modification in telomeres, partially due to their high G-content [91]. 8-oxoguanine lesions are particularly difficult to repair by the 8-oxoguanine glycosylase OGG1. Such ROS-induced lesions can be further translated into single- and double-strand breaks, or result in replicative stress, eventually all leading to telomere damage and dysfunction, including shortening. Moreover, the presence of unrepaired 8-oxoguanines inhibits the binding of TRF1 and TRF2 [92], and can also impair the recruitment of telomerase, when they are localized at the overhang [93].
Telomeric DNA strand breaks that accumulate during the interphase, get translated into TS in the next round of DNA replication [94]. This process might be of high importance for cells in vivo that divide just sporadically or as a result of internal or external stimuli, for example, during high regeneration phases after injury. In other words, a higher TS would be detected when more cells of a tissue that have accumulated DNA damage during an extended stationary phase divide and translate their accumulated strand breaks into TS. In that way, these oxidative lesions as well as DNA breaks are able to result in telomere dysfunction as well as shortening. See more details about oxidative stress and its influence on TL and TS in the next section.
In addition to the gradual TS during DNA replication, a process called “telomere trimming” has been described that represents an additional mechanism of telomere length control contributing to normal telomere dynamics [95]. In this process, a specific protein called TZAP (telomeric zinc finger-associated protein) binds to rather long telomeres when there are low amounts of shelterin components TRF1 and TRF2 present resulting in a rapid deletion of telomere repeats [96]. Telomere trimming involves the resolution of intermediate structures that occur during recombination, which shortens telomeres and releases extrachromosomal telomeric DNA [95]. The occurrence of such extrachromosomal telomeric sequences might potentially influence TL measurement methods. Importantly, this process also sets an upper limit for telomere length and thereby contributes to a length equilibrium setpoint in cells that harbor a telomere elongation mechanism [95]. Telomere trimming is controlled by XRCC3 and Nbs1, homologous recombination proteins that generate single-stranded C-rich telomeric DNA and double-stranded telomeric circular DNA (T-circles), respectively. These two structures are markers for active "telomere trimming" and have been found in tumors, healthy brain tissue [97] as well as in stem cells (human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)) where the process seems to contribute to normal telomere homeostasis [98]. Specific techniques such as quantitative fluorescence in situ hybridization (qFISH) are best suited to detect such sporadic loss of large telomere sequences [99].
In addition, an accumulation of TERRA molecules at telomeres can also interfere with the replication of telomeres and result in a sudden loss of telomeric sequences [100].
Moreover, replication forks can get stalled at telomeres and consequently collapse which can result in DNA breaks. These breaks should be either repaired by recombination events while otherwise long regions of telomeric DNA can get lost [101].
As already mentioned above, telomere shortening is not even during lifetime and highest during the first 1-2 years of life [84]. In addition, there is also a high heterogeneity in TL at different chromosomes [19] which might even display a chromosome-specific shortening rate [20].
Finally, inherited mutations in telomerase as well as telomere-related genes in conditions known as telomeropathies result in lower TA and decreased TL from early embryogenesis on, are responsible for the early onset of degenerative conditions in highly proliferative tissues such as the bone marrow and a premature death [102].
4. Telomeres and Oxidative Stress
Oxidative stress is an important factor responsible for telomere shortening in addition to the ERP in various cell types including T-lymphocytes and fibroblasts [103,104,105]. Telomeres are preferentially susceptible to oxidative damage caused by the high content in guanine-rich sequences in telomeres which are more vulnerable to damage than other nucleotides [106]. Oxidative stress and inflammation as well as antioxidant enzymes could also be, in addition to inherited TL, important factors contributing to inter-individual TL differences.
Telomere length and dynamics are not just an indicator for senescence and aging, but also for various physiological and psychological stress factors. In that way telomeres seem to constitute an important biomarker of an environmental exposure as well as various lifestyle factors such as nutrition, toxins and exercise, to name just a few. The influence of oxidative stress also proofs the initial claim of telomeres functioning as a mitotic clock wrong when initially only their role in replicative senescence was known. Today it is clear that telomeres are under a wide variety of influences, from genetic ones via DNA replication and inheritance of certain TL (please see more details below) to the role of telomerase activity, TERRA and environmental factors.
Oxidative stress is mainly derived from reactive oxygen species (ROS) produced at different cellular sites and by different pathways, predominantly from mitochondria and mitochondrial dysfunction [89], but also other ROS sources such as NOXes can be involved. ROS directly target DNA and can modify it in different ways. It can induce direct breaks such as single strand breaks into the DNA or modify nucleotides the DNA consist of such as the most common 8oxodG.
Oxidative stress can causally damage telomeres, but both factors are also related to each other in a two-directional feed-forward loop [107,108]. At the same time, a large number of antioxidant enzymes and factors exists that is able to counteract cellular oxidative stress via a number of mechanisms. Consequently, oxidative stress is always a balance between generated pro-oxidants and their counteracting antioxidants. Interventional experiments have demonstrated that a decrease in ROS due to antioxidants is able to counteract downstream telomere shortening [104,105] while not ameliorating the upstream mitochondrial dysfunction responsible for the increased ROS generation during senescence, aging or specific diseases such as mitochondriopathies which also show an increased TS [109]. Oxidative stress can be viewed as a biological effector of metabolic processes as well as endocrine stress-related hormones and inflammation.
Importantly, ROS can damage telomeres in mitotic as well as post-mitotic (PM) cells (see below). Telomeric damage does either manifest as single-stranded DNA strand breaks [38,110] or as oxidative modifications, predominantly 8oxodG [91] which can later also be transferred into strand breaks in dividing cells. In in vitro experiments increased oxidative stress was able to accelerate TS up to 6-fold resulting in an earlier onset of replicative senescence of human primary fibroblasts [2,27] while the exact amount can vary by cell type. DNA strand breaks which accumulate due to oxidative stress while a cell is quiescent, will in mitotic cells during the next round of DNA replication translate into telomere shortening [94].
At the same time the TS rate will be also modified by the individual cellular antioxidant capacity (AOC) and is thereby genetically determined [2,77]. Von Zglinicki and colleagues found that different cell types of the same individual, PBMCs and skin fibroblasts, have similar antioxidant capacities [2]. This means that individuals with a high AOC are less sensitive against oxidative stress than those who have a low antioxidant capacity which renders their cells and tissues more susceptible to oxidative stress. Other genetic studies confirmed this finding, at least indirectly. This correlation makes TL and TSR good parameters to determine biological opposed to chronological age, the aging process and a possible susceptibility for aging-related diseases at the individual level. However, such a relationship can only rarely be detected in cross-sectional studies due to large inter-individual differences in TL of 5-6 kb [111] while a better way to include individual TS rates as read-outs for a participant’s AOC are longitudinal studies where each individual’s telomere trajectory will be monitored and analysed as already performed in some studies where it predicted lifespan and mortality [23,112]. Intriguingly, a very recent study from 2025 revealed that oxidative stress also induces internal DNA loops, TRF1 dissociation, and TRF2-dependent R-loops at telomeres [110].
Importantly, it has been shown consistently, that a number of early life stresses (intrauterine, prenatal and early post-natal) may contribute to short TL during childhood and therefore predispose subjects to poor health in later life [99,113].
The influence of oxidative stress on TL in vivo was recently confirmed in different meta-analyses [114,115]. Importantly, the latter meta-analysis found that the particular telomere length measurement method was an important factor for a successful correlation of oxidative stress with telomere dynamics (TL and TS) with the TRF method using the rather laborious Southern blot technique giving the best results. In contrast, most human studies rather use the much quicker qPCR method from [3]. Interestingly, the correlation between sensitivity to oxidative stress and TS seems to be more pronounced in faster aging organisms such as birds. Another aspect to consider is a possible variation of oxidative stress between different organs and tissues [116] which might depend on the specific regenerative capacity in these tissues and organs.
Another intriguing source for oxidative stress seems to be psychological stress. Epel and colleagues demonstrated that for the first time in 2004 for care-giving mothers who showed shorter TL and lower TA in their PBMCs [117]. A possible explanation was that such psychological stress gets converted into physiological and oxidative stress in the body. Glucocorticoids could play a role in that process as well. For example, TA levels can get reduced in vitro due to glucocorticoid treatment in T-lymphocytes [118]. Such stress hormones are able to modulate metabolic rate and mitochondrial activity [119] and increase the expression of pro-inflammatory genes [120], thereby promoting inflammatory processes which will be described in more details later. Useful meta-analyses, including animal experiments, have recently confirmed an association between short TL and various types of stresses, both physiological and psychological types [119,121,122].
There is a substantial difference regarding telomeres in dividing/mitotic compared to post-mitotic cells. While the former shorten their telomeres due to the ERP (see above) with oxidative stress being a modulating factor, the latter as non-dividing cells, do not shorten their TL but can still accumulate DNA damage due to oxidative stress and inflammation which can activate the DNA damage response (DDR) and result in senescence with downstream features such as SASP. However, not all markers of cellular senescence in dividing, mitotic cells have been also clearly identified in PM cells, for example sen-β-gal (senescence-associated beta-galactosidase) [123] which seems to be rather tissue-specific. Such consideration about differences in telomere-induced damage in dividing and non-dividing cells is instrumental when trying to establish an association between TL in blood cells such as mitotic leukocytes and various, mainly aging-related diseases which are very often occur in non-blood cells and frequently even in post-mitotic tissues such as brain (which consists of postmitotic neurons and mitotic glia cells), heart, skeletal muscle, fat etc. [124,125] while these tissues still contain division-competent stem cells. It has been demonstrated that under normal physiological conditions, persistent telomeric damage accumulates with age, for example in the gut and liver of mice [36], as well as in neurons and hepatocytes of primates [37], irrespective of telomere length. Intriguingly, it has been recently suggested that telomere-related senescence in post-mitotic cells could be a major driver of the aging process in vivo [30].
5. Induction of Senescence or Apoptosis by Short or Dysfunctional Telomeres
As already outlined above, telomeres shorten during repeated rounds of replication in dividing cells and also accumulate damage due to increased oxidative stress. In mammalian fibroblasts telomeres reach a critically short length at around 5 kb when checkpoints are induced and a DNA damage response is initiated via DNA damage-associated proteins such as γH2A.X or p53BP1 which bind to the damaged areas of the DNA forming DNA damage foci [126]. These foci can be employed to determine the degree of telomeric damage using a technique called Telo-FISH combining an antibody against one of these proteins and a PNA-labelled telomeric probe which indicates the localization of the damage to the telomeres (TIFs, TAFs) and can be in that way separated from general genomic DNA damage [36,37,127] (see Figure 4). The same DDR is induced in postmitotic cells when persistent DNA damage is accumulated in telomeres. For example, it was shown in differentiated human adipocytes in vitro that telomeres accumulate 10 times more DNA damage than the remaining genome [128].
Figure 4 Telomere signals and telomeric damage. A: Telomere signals (pink) on interphase nuclei and metaphase spreads. B: A telomeric PNA probe and a DNA damage antibody (γH2A.X) where overlayed on a DAPI-stained nucleus and the merged yellow signal shows a telomeric damage (TIF or TAF).
As already described above, telomeres accumulate DNA damage preferentially forming a sentinel due to their high sensitivity against oxidative stress, which activates the DDR and drives cells to go into senescence or apoptosis. These alternative outcomes often depend on the specific cell type as well as the severity of the induced damage. When telomeres reach a critically short length or have accumulated persistent DNA damage, cells with intact DDR checkpoints enter a cell cycle arrest and, since telomeric DNA repair is rather limited, eventually senescence or apoptosis are induced via p53 and p21 as well as additional, pathway-specific genes.
While the majority of the DNA damage in other parts of the genome gets eventually repaired by different repair mechanisms, DNA damage at telomeres causes a persistent type of DNA damage that leads to a DDR and eventually, senescence or apoptosis [36,37,129]. For example, a study showed an increase of telomeric dysfunction as a read-out for cellular senescence in baboon skin which increased exponentially up to 15% of cells at very high age [130]. The DDR is a signaling pathway which involves a cascade of kinases (ATM/ATR), mediators (Chk1, Chk2) and finally activates the tumor suppressor gene p53 and the downstream p21 stress-response gene [126].
Consequently, some scientists speculate that the accumulation of unrepaired telomeric damage could be the main source of telomere-driven senescence rather than telomere shortening due to the ERP [30,131]. This suggestion makes sense considering that most cells and tissues do not have a high division rate in vivo.
When some of the important checkpoint genes, like, for example p53, are, however mutated or inactivated, then TL can further shorten resulting in telomere-to-telomere fusions, anaphase bridges and genomic instability. While most of these cells die after such crisis, some cells re-activate TA which caps and maintains stable telomeres which, together with the activation of oncogenes and inactivation of tumor suppressors, can develop into malignant lesions and eventually, tumor cells [132].
In addition to critically short telomeres also the collapse of the telomere structure can cause a DNA damage response and eventually result in senescence or apoptosis induction. As a reason for such change in telomere structure the dysfunction or imbalance of the telomere binding shelterin complex has been described. For example, overexpression of TRF2 increased the rate of telomere shortening in somatic cells, but without accelerating the senescence process. The mechanism behind this is a reduction of the senescence setpoint, which is the telomere length at which senescence sets in, from 7 to 4 kilobases and thereby delayed senescence [133]. The authors demonstrated that overexpressed TRF2 protected critically short telomeres from end-to-end fusions. The authors concluded that more important than absolute TL is the protection of shortened telomeres, also known as telomere capping [86]. In addition, also other shelterin components such as human Rap1 which binds to TRF2, are involved in TL regulation. Importantly, hRap seems to contribute to TL distribution and heterogeneity [134]. In addition, it has been suggested that the frequency of the shortest telomere(s) determines the time point when a cell enters senescence [21,22,135].
Recently another mechanism has been described where telomere damage and dysfunction via the activation of a DDR activate the cytosolic DNA-sensing pathway and eventually induce premature senescence independently of TL [136]. This mechanism involves the sensing of telomeric and chromatin fragments via the cGas and STING system resulting in an innate immune response [137].
6. Characteristics of the Senescent Phenotype and Its Contribution to the Aging Process
Senescence is defined as an irreversible cell cycle arrest (at least in mitotic cells, not in PM cells) and the activation of p53/p21 and p16/RB pathways and includes DNA damage together with the DNA damage response, mitochondrial dysfunction, striking morphological changes as well as epigenetic alterations. Interestingly, Lopez-Otin and co-authors suggested that cellular senescence as a tumor-suppressor mechanisms could be viewed as a beneficial compensatory response to excessive DNA damage that, however, can get deleterious and promote the aging process when there are too many senescent cells in a tissue which hampers regenerative ability [1]. Aging is a process of declining physiological and cellular functions including a decreased fertility and reproduction [138]. The process has been demonstrated to involve a complex interaction of multiple pathways that have been named “hallmarks of aging”. These mainly include genomic instability, telomere shortening, epigenetic alterations, impaired protein homeostasis (including the autophagy-lysosomal system and proteasomal degradation), dysregulated nutrient sensing including insulin, mTOR and AMPK signaling, mitochondrial dysfunction including ROS generation and deregulated biogenesis, cellular senescence, decrease of stem cell functionality due to different mechanisms as well as changed intercellular communication [1] which were further updated recently [139].
In mitotic cells the most widely recognized feature of senescence is the irreversible cell cycle arrest. This is often caused by TS eventually reaching a critically short TL and inducing the DDR that has been described in the previous heading. Frequently, senescence is also viewed as a stress response [140]. In contrast, PM cells which do not divide, don’t have this particular feature of telomere shortening while accumulating telomeric damage which might be causally involved in the development of the other senescence-related characteristics [141] and is also viewed as a stress-response [30]. Senescence is a heterogenic phenotype that can be triggered by both, external and internal factors. Here only internal factors such as telomeres and mitochondrial dysfunction will be briefly described while external factors drive primarily stress-induced cellular senescence (SIPS) that is telomere length-independent [34] but might also involve telomeric damage. Importantly, it has to be kept in mind that the continuous process of TS is not identical with replicative senescence since the latter is just the final step of a life-long process until telomeres reach a critically short length which results in telomere uncapping and the induction of the DDR. However, this process has been mainly studied in cell culture experiments in vitro, while there are not many data available about how many cells/cell types really reach replicative senescence in vivo. An exception are T-lymphocytes which are responsible for immuno-senescence in older individuals and the fact that many older people eventually succumb to infections due to the exhaustion of their immune system, unable to cope with those [142,143]. Immuno-senescence is thereby a major obstacle to increase human longevity. Senescence and telomeres in immune cells will be described in more details later.
In addition to critically short telomeres, pathways like p53/p21 and p16Ink4a/retinoblastoma protein (RB) are causally involved in the establishment of an irreversible growth arrest and senescent cells [144]. Continuously high levels of the cell cycle inhibitor p16 are instrumental for maintaining senescence [145]. An important role in the senescence phenotype is related to mitochondrial dysfunction including the generation and release of ROS which can further damage telomeres and genomic DNA in a feed-forward loop [107]. The study from Passos and co-authors found that a telomere-induced DNA damage response (DDR) triggers a dynamic feedback loop with the long-time maintenance of cellular senescence. This loop is responsible for the long-term activation of the checkpoint factor p21 that induces mitochondrial dysfunction and ROS generation via a serial signaling through GADD45 onto TGFβ. This ROS can then induce further damage to telomeres and other genomic regions [107]. Other characteristics of senescence are high p16Ink4a levels, and the senescence-associated secretory phenotype (SASP) [146,147,148]. In particular, the secretion of numerous biologically active factors via the SASP can contribute to physiological and pathological consequences in organisms. The secretome of senescent cells is rather complex, but also variable between cells and induction conditions. It includes a broad range of cytokines, chemokines and proteases, such as MMPs (metalloproteinases) and VEGFs, to name just a few, that contribute to both, pro- and anti-inflammatory signaling [147,148,149]. In particular, NF-κB plays an important role for a pro-inflammatory phenotype with IL-1 being upstream and IL-6 and IL-8 being important downstream effectors [148,149]. While the SASP is controlled at various levels such as transcription and autocrine factors, a persistent DDR from telomeric damage appears to be critical for regulation of the SASP while other factors involved in senescence such as p16 that functions independent of the DDR are unable to induce the SASP [146]. At the same time, IL-1 and TGFβ are able to promote oxidative stress and DNA damage in a feed-back loop [150]. Moreover, the NF-κB signaling pathway was identified as a key regulator of SASP which can be stimulated by DNA damage [151]. Downstream effects of the SASP are context-dependent. They can be anti-tumorigenic, pro-tumorigenic, immunomodulatory or modulate the tissue microenvironment in a paracrine manner [152].
However, the senescence phenotype can also be heterogenous and dependent on the inducing trigger. For example, oncogene-induced senescence (OIS) is characterized by heterochromatic foci (SAHF) [29] while these foci occur less prominently in other senescence types with, for example, cells in replicative senescence possessing only rather few of such foci [153]. SAHF consist of repressive epigenetic marks, such as methylated H3K9, HP1, and macroH2A and persistently suppress E2F-target genes to maintain a stable senescence state, predominantly during OIS [29].
While it is important to separate cellular senescence at the levels of cells from the aging process of a whole organism, both processes are still tightly connected. Senescent cells accumulate during the aging process in an organism where they occupy cellular niches and secrete pro-inflammatory cytokines via the SASP, having an adverse effect on the tissues and organs they reside in due to their bystander effect which includes the secretion of reactive oxygen species (ROS) [129] and soluble pro-inflammatory molecules from the SASP that target other cells in the vicinity and thereby accelerate the aging process of the whole organism and contribute to aging-related diseases and morbidity [154]. The authors suggest that the abundance of senescent cells in vivo might predict the biological age which is different from chronological age. In contrast, removing senescent cells from the body has a rejuvenating effect and can even reverse several characteristics of aging as well as age-related diseases. Pioneering experiments have been performed with either genetic constructs (INK-ATTAC) in order to induce apoptosis in p16Ink4a-expressing cells in model-and wild type mice by injection of the drug AP20187 which showed that the removal of senescent cells even in old mice is able to rejuvenate them, to extend lifespan as well as health span and to delay and attenuate the progression of various aging-related diseases [155,156]. The same group demonstrated with the same genetic system or various senolytic agents that removing senescent glia-cells from different mouse models of Alzheimer’s disease (AD) greatly improved the disease phenotype by decreasing tau- and amyloid pathology [157,158]. Meanwhile, there have been many similar studies been published showing the great potential of the removal of senescent cells from the mammalian body with different senolytics which are mainly based on treating the SASP component of senescent cells as an alternative to treating aging-related diseases [159]. These studies demonstrate the tight link between senescence and aging which can be delayed by removing senescent cells. Their results as well as future clinical implications are summarized in various recent papers and reviews [160,161,162,163].
7. Genetic Components (Inheritance) of TL
Telomere length (TL) is highly variable across adult humans [164]. Evidence suggests that this variation, as measured in leukocytes, is already established at birth. The inheritance of TL might explain the high variability of leukocyte TL (LTL) between individuals in cross-sectional studies [111,165,166]. Various of these studies found that around 60% of inter-individual variation in LTL at baseline and 30% of the age-dependent TS seem to be heritable [166,167]. This result seems to suggest that during adult life TSR can be modified to a certain degree due to environmental and lifestyle influences up to 70%. However, it also does not exclude earlier suggestions that age-related changes in TL seem to be dependent on TL at baseline early during development [26]. Telomere shortening is highest during the first two years of life [83,84], while the pattern of chromosome-specific variations in telomere length might be possibly maintained throughout life [168].
Both, TL at birth as well as the individual TS rates seem to be determined, at least partially, by inheritance and genetic predisposition. For example, TL in PBMCs (peripheral blood monocytes) of young dizygotic and monozygotic twins showed around 78% heritability [165,169]. This observation is reflected in a wide spread of TL over large age-ranges as demonstrated in numerous cross-sectional studies [2,170]. Both parents and their age have an influence on their offspring’s TL [171,172]. Since sperm TL does not decrease with age but even increases [173,174], most likely due to high activity of telomerase in testis [175,176], offspring from older fathers tend to have longer telomeres [174,177,178,179]. In contrast, TL does not seem to depend on maternal age at the time of birth in humans [180] while it has been demonstrated in other species such as birds [181].
While oxidative stress can accelerate telomere shortening (see earlier heading), the shortening rate also depends on the antioxidant capacity of the cell and might therefore be genetically determined [2,77]. It has been demonstrated that different cell types of the same individual (for example PBMCs and skin fibroblasts) have corresponding antioxidant capacities despite differing absolute TL possibly reflecting replicative history [2]. With other words: cells with a high antioxidant capacity are less sensitive against oxidative stress than cells with a low antioxidant capacity that are more susceptible to oxidative stress. All these properties were thought to make telomeres good biomarkers of aging which is today more and more disputed with epigenetic markers getting a better association with the aging process [182]. Some authors claim that genetic influences might be less pronounced for TS than for TL, at least in birds [183].
Importantly, absolute TL and the rate of TS can also vary between different tissues as the study by Demanelis and colleagues has demonstrated [184]. The authors analyzed the variability of genetic determinants of TL across more than 25 different post-mortem human tissues from nearly 1000 individuals. Their study demonstrated a large genetic variability across these different tissue types which needs to be considered when interpreting the results of various epidemiological studies. The authors found a good correlation of relative TL between tissues from the same donor with those from lymphocytes being the shortest, but in general establishing itself as a good proxy for that individual’s TL. The study also found that the influence of age was more pronounced in those tissues with the shortest TL such as PBMCs. However, there was a large variation between individuals, as previously described in other studies [81,185,186,187,188], rendering the results from cross-sectional studies rather unreliable by obstructing the situation in each study participant since TL can also greatly fluctuate over time in blood cells [189]. Intriguingly, there was a strong influence of genetic ancestry on general TL with those of African ancestry having significantly longer telomeres in most tissues [184], making studies with participants of different ancestral background difficult to interpret. In addition, the study also found the highest correlation to age in some tissues, prominently PBMCs, while not in other tissues such as cerebellum and muscle as PM tissue, confirming their suitability for use as a reference tissue for determining specific TS in blood cells excluding inherited TL differences with a method described below. Intriguingly, the study also described a rather strong mediating effect of age-related TL on the expression of age-related genes which means that there might be a direct influence of short TL on an aging phenotype.
There is still an ongoing dispute in the telomere field whether it is rather the LTL at birth that pre-determines an individual to certain diseases such as CVD (cardio-vascular disease) or whether oxidative stress and inflammation might be the more important parameters [164]. Nevertheless, the influence of different lifestyle and environmental factors on the modulation of TL and TS argues at least partially for the significance of such additional factors.
A method to consider the intra-individual variation of inherited TL was proposed by A. Aviv’s group. They published an intriguing paper where they used a canine model to discriminate TL and TS in lymphocytes, the most frequently used tissue for human studies, and several mostly postmitotic tissues such as muscle and fat in order to establish a longitudinal TL dynamics in blood lymphocytes [185]. Leukocyte TL depends to a large extend on that of hematopoietic stem cells [83,190]. A study found a high amount of cell divisions in hematopoietic stem cells (HSCs) of 17 within the first year of life which decreases down to around 2,5 per year during childhood and finally only amounts of 0.6 times per year in adulthood [83]. The study from Benetos and co-authors [185], similar to various human studies, has detected differences of 4-6 kb at birth as well as during the lifetime of individuals which is a large hindrance in correlating blood TL to different diseases in cross-sectional studies often leading to contradictive results. The approach from that study considers both, the initial TL at birth in muscle which can depend on both parents, as well as the individual telomere shortening rates (TSR) of the different organs and tissues including lymphocyte TL by determining the difference of both tissues during aging for around 4 years. As expected, their model found no change in muscle and fat TL during this time-period while TL in lymphocytes declined over time with increasing age corresponding to a 40 bp decrease per month [185]. In contrast, taking the difference between lymphocyte and muscle tissues for each individuum generated a much steeper decline of the regression line in TL corresponding to 52 bp per month (see Figure 5). In general, dogs have a much higher TSR than humans [191]. The study found that lymphocyte TL was a strong predictor of average lifespan in different dog breeds [185].
Figure 5 Principal results from study [185]. While telomeres shorten during life in PBMCs from blood (B, C), TL from a post-mitotic (PM) tissue such as muscle stays more or less constant and reflects the inherited initial TL from early development (A, C). Subtracting the inherited PM-TL from that of the PBMC TL generates a much steeper slope and now better reflects the telomere shortening (TS) during adult life [185]. This “blood-and-muscle method” was later also applied successfully in human studies [187,188].
Similar findings of matching relative telomere lengths in different tissues and organs of the same individual have been reported in humans [2,184,187,192]. Consequently, the model from Benetos and coworkers [185] subtracting TL of muscle tissue from leukocyte TL generates much more realistic leukocyte TL dynamics data because they are based on the individual telomere dynamics rather than across a population as in most cross-sectional studies. Such an inter-individual variation in age–dependent leukocyte telomere shortening can then be used to correlate it to other physiological parameters including aging, longevity or different types of aging-related diseases. This cannot be accomplished based on the cross-sectional evaluation of leukocyte TL where in the conventional cross-sectional way, age could explain only 6% of the TL changes over the 3 years of dog life, while in the subtractive method 42% of TL changes in lymphocytes could be explained by age and there was an around 7 times higher specificity for the used differential method [185]. This, however, would mean a much higher amount of work for epidemiological studies including biopsies of PM tissues from people which is much more invasive than just taking blood from participants and will therefore most likely not gain easy access into clinical practice. However, for academic and animal studies, such an approach might be feasible and should be considered for future research.
The intriguing fact that individuals seem to maintain their relative LTLs such as short or long LTL, compared to other subjects of similar age suggests that this specific long and short TL might also contribute to the biological age of this person as well as their susceptibility to different diseases [81,186]. Extending the methods from Benetos and co-authors [185] described above, the group applied the “blood-and-muscle method” also to human adults [187] as well as fetuses and newborns [188]. Both studies found that relative TL between the two tissues correlated well, being either short, average or long. The latter study also found that already in fetuses there was a lower TL in leukocytes than muscle meaning that the original common, inherited TL for both tissues must have occurred even earlier in development, presumably in early gestation before TA is switched-off in most human somatic tissues [193,194]. Interestingly, the study also found an inverse correlation between body mass index (BMI) and TL in both tissues, possibly due to higher cell replication during growth or increased oxidative stress in fat tissue. The authors emphasize that the inter-individual variation in TL exceeds that of intra-individual differences up to three times while the gap between LTL and TL in muscle tissue widens throughout life due to higher cell division amounts in leukocytes than muscle or other PM tissues. The important conclusion from these studies is that variation in TL between individuals is inherited and thus appears from the earliest development onwards [195]. The findings also suggest that these differences might be causally involved in the susceptibility of individuals with short or long telomeres to aging-related diseases such as CVD and cancer, respectively [188]. This causality was further explored and confirmed by large genetic GWAS studies that generated genetic risk scores for different diseases [196,197].
8. The Role of Telomerase for TL in Lymphocytes
Most human studies use TL in white blood cells, also called PBMCs or leukocytes consisting of lymphocytes (T- and B-cells, natural killer (NK) cells, monocytes and granulocytes) since they are accessible by a non-invasive blood draw. These blood cells are unique since they are under the influence of other organs and processes such as inflammation as well as external factors via nutrition, environmental factors and stressors. However, telomere length in blood cells is specifically influenced by the potential presence of telomerase activity (TA) in lymphocytes which can be upregulated upon stimulation and is highly regulated at a physiological level while resting lymphocytes show hardly any TA [198,199,200]. Unfortunately, TA regulation in blood cells is not well understood and has not been extensively examined yet. For example, contradicting data exist on the influence of oxidative stress and inflammation on TA in immune cells which might also depend on the stimulus, time and severity of stress as well as the particular cell type. For example, TA decreases under chronic oxidative stress in mouse splenocytes in vitro [201]. In contrast, TA and its protein subunit hTERT (human telomerase reverse transcriptase) are stimulated in human macrophages upon oxidative and atherosclerotic challenges in vitro and in vivo during the formation of atherosclerotic lesions [202].
Similar results were found under acute or chronic psychological stress where TA in PBMCs either increased or decreased, respectively [117,203]. These studies demonstrated that while a chronic decrease in TA was accompanied by shorter average TL, the acute increase in TA was accompanied by increases in the stress hormone level of cortisol but might not have influenced TL. Recently, Guillen-Parra and colleagues [204] demonstrated in a longitudinal study the role of TA and stress on lymphocyte TL. The study found that both, chronic stress and lower mitochondrial capacity independently influenced a decrease in telomerase activity over the 9 months of observation. Importantly, changes in telomerase activity directly impacted telomere length.
Telomerase activity is highly expressed in hematopoietic stem cells [205], during B-cell differentiation in the germinal center and during T-cell development in the thymus [206,207,208] but is low or undetectable in resting lymphocytes [198]. Average TL in PBMCs most likely reflects the hematopoietic stem cell (HSC) compartment in individuals. T-lymphocytes are generated in the bone marrow and afterwards migrate to the thymus for maturation. Naïve T lymphocytes circulate between blood and other lymphoid organs before they encounter their specific antigen. After immune response, a small fraction remains as memory T-cells, while the majority dies. During the process from naïve T to effector and memory T cells they undergo various adaptive proliferative, metabolic and oxidative processes.
Upon activation, T-cells and B-cells rapidly up-regulate telomerase activity which partially compensates for lost telomere sequences in dividing T-cells. Importantly, telomerase is able to extend the replicative lifespan of T-cells via ectopic expression of TERT [209]. In contrast, telomeropaties with inherited mutations in telomerase or telomere components result in shortened telomeres already during early development leading to a functional loss in proliferating tissues and organs including the bone marrow [210].
Telomerase activity is highly regulated during human lymphocyte development, differentiation, and activation [206,211,212,213,214]. Intriguingly, independent of the status of telomerase activity both, hTERT transcripts and protein are present in all lymphocyte subsets isolated from thymus and peripheral blood, presumably in order to quickly activate TA [215]. Unstimulated T-lymphocytes and natural killer (NK) cells contain cytoplasmic telomerase protein hTERT which enters the nucleus only upon activation and phosphorylation [215]. However, this different biological behavior poses the problem that the measurement of hTERT expression or even the measurement of telomerase enzymatic activity in a cell-free assay (TRAP) that uses whole cell lysates might give results which are different from the in vivo situation where the telomerase complex is only assembled when TERT moves from the cytoplasm to the nucleus and telomerase can access telomeres for their extension. Various stimuli such as IL-2 promote hTERT transcription and telomerase activation via Akt-phosphorylation, hsp90 and mTOR [216] as well as the transport of TERT from the cytoplasm to the nucleus [217,218].
However, TL still shortens during aging or disease in immune cells, presumably due to their high replicative activity. The high replication number might be the reason why TL is usually shorter in PBMCs compared to other human somatic cell types [2,185,188,192]. In addition, it had been demonstrated previously, that limited amounts of TA, like in PBMCs, might work predominantly, but not exclusively, at the shortest telomeres while the longest telomeres shorten continuously [219]. In general, studies have shown that to better maintain LTL via higher TA might be a prerequisite for a longer life since that feature has been described in PBMCs of healthy centenarians compared to unhealthy ones [220,221].
While moderate levels of mitochondrial ROS are required for the activation, differentiation, and effector functions of lymphocytes, increased levels of inflammation and oxidative stress downregulate TA and compromise T-cell proliferation and activation [201,222].
Importantly, TL varies in different T-cell subsets with higher TA and longer telomeres present in naïve T-cells compared with memory T-cells [142,200,223]. Telomerase activity is an important prerequisite for the proliferative capacity of lymphocytes which can decline during immuno-senescence corresponding to short TL and associated with the aging process [142].
Genetic studies on human inherited diseases caused by mutations in genes encoding telomere and telomerase components (telomeropathies) demonstrated that a low level of telomerase activity is a determining factor for short leukocyte telomere length [224]. Likewise, clinical studies showed that high levels of senescent CD8+ T cells strongly correlated with several negative physiological parameters and increased amounts pro-inflammatory cytokines [222]. Cytokines and hormones regulate TA either up or down which might impact TL as well [203,216,225,226]. Some studies suggest that lifestyle activities such as exercise might impact health via increase in leukocyte TA and TL [227].
Lin and colleagues systematically analyzed age-related changes in lymphocyte TL in vivo in a longitudinal study over a 12-year period [200]. The study had more than 200 participants from the Baltimore Longitudinal Study of Aging with an age range of 20-90 years. The authors found that TS in B- and T- lymphocytes depends on 3 factors: TA, changes in lymphocyte subsets and general health conditions of the donors. As expected, TA decreased over time in T-cells which corresponds well with the results from Plunkett and colleagues [142] who found a declining TERT phosphorylation by Akt in CD4+ T-cells as a possible underlying mechanism. However, surprisingly, only around a third of participants really decreased their lymphocyte TL during the 12 years of analysis, while 50% of TLs rather stayed constant and 10% even increasing their TL. This finding already shows that the situation with TL in lymphocyte is far from being straight forward, but rather more complex in accordance with results by others who demonstrated that TL in blood cells might show an undulating/oscillating TL over time, possibly correlating to changing TA levels, and thus be rather dynamic at the individual level [189]. The study by Lin and co-workers also demonstrated that TS in lymphocytes might rather slow down over time with age and might, in addition, depend on many different parameters, such as chronic inflammation (see heading below), acute diseases or environmental exposures, lifestyle as well as the change in lymphocyte subsets over time.
Leukocytes originate from HSCs and differentiate into different subsets, such as lymphocytes, monocytes and granulocytes (neutrophils, basophils and eosinophils). The cellular composition of PBMCs can be rather variable even in healthy subjects and even more so during diseases due to varying levels of oxidative stress and inflammation. Similarly, telomere length differs between leukocyte subsets [200,228]. Lin and coworkers demonstrated that telomere length in T-cells correlated well with the fraction of naïve T-cells in the total T-cell pool and inversely with the fraction of senescent CD28− T-cells [200]. This finding strongly demonstrates that the composition of T-cell subsets is a major factor for changes in telomere length in T-cells with increasing age. A similar finding was also published by Deelen and colleagues who found that the variation in donor TL was associated with differences in leukocyte subset composition, such as lymphocyte, neutrophil and basophil counts [170]. This result strongly suggests that mean TL is influenced by the composition of the different leukocyte subsets.
It also emphasizes the importance of analyzing changes in lymphocyte composition either due to aging or diseases for epidemiological studies correlating TL to various diseases and conditions. In summary, TL in leukocytes is influences by various factors such as TA, subset composition as well as the replicative history of lymphocytes while in short-lived granulocytes they rather reflect the inherited TL from HSCs. Nevertheless, a study has described an existing synchrony between leukocyte subsets throughout human lifespan [229]. This means that individuals with relatively long/short TL in one leukocyte subset have a similar long/short TL in other subsets which again clearly emphasizes the genetic component of TL.
Interestingly, it seems that the large variability of TL might decrease at higher age in T-cells and memory T-cells of healthy older people around 85 years [230]. Surprisingly, the older participants had not particularly long telomeres, but shorter TL compared to middle-aged subjects. In the light of the known association of short TL with degenerative diseases and long TL with cancer, the authors suggest that possibly a selection for an optimal, average TL might be the underlying mechanism. This result, yet another time, underlines that absolute values of TL might not be as good biomarkers as previously thought and that an interpretation of TL in cross-sectional studies without knowing telomere dynamics of individuals, should be done rather cautiously.
A completely new mechanism on telomere maintenance in T-cells independently of TA was recently reported by Lanna and colleagues [231]. The study demonstrated that some T-cell types including memory cells were able to extend their telomeres via the acquisition of telomere-carrying vesicles from antigen-presenting cells (APCs). When T-cells got in contact at immunological synapses, the APCs degraded shelterin at their telomeres which were trimmed by the factor TZAP and then transferred to extracellular vesicles. Those used the recombination factor Rad51 to perform a telomere fusion with T-cell telomeres and thereby lengthened the latter for an average of 3 kbp which is much longer than TA could achieve. In that intriguing manner, these T-cells got rejuvenated and stem-cell like by preventing senescence and enabling them for long-time clonal division and thus contributed to an immune protection. It will be interesting to see in future studies, how common this mechanism is in preventing immuno-senescence of T-lymphocytes or whether it might rather promote the development of haematological cancers [232].
9. Inflammation and TL
Inflammation and oxidative stress were identified as important processes causing and accelerating telomere shortening and damage as well as being associated with various diseases such as CVD [233]. The influence of inflammation on TL and TS are particularly important for the aging process which is characterized by an increased chronic inflammation, often also called “inflammaging” [234].
Inflammation can have various underlying causes such as senescent lymphocytes and immuno-senescence, tissue damage, macrophage activation and the induction of pro-inflammatory cytokines. In the immune system inflammation can induce leukocyte proliferation in the bone marrow resulting in more naïve lymphocytes with long TL while mature lymphocytes have much shorter TL [229].
In addition, cellular senescence has a strong inflammatory component through the senescence-associated secretory phenotype (SASP) which contains many different pro- and a few anti-inflammatory cytokines and chemokines [235]. In that way, senescent immune cells can, for example, contribute to the “inflammaging” phenotype, in particular, to a systemic inflammation [236]. Finally, inflammation underlies many human diseases. However, the role of TA and inflammation remains contradictory. While in patients with metabolic syndrome TA was increased due to inflammation in PBMCs [237], there was a drastic downregulation of TA in PBMCs of patients with an acute ST elevation myocardial infarction (STEMI) which is associated with high inflammation while an improvement of the condition after 3 months resulted in normal TA levels [238]. A possible link between inflammatory signaling and the activation of telomerase in leukocytes might be the mediation of nuclear translocation of the TERT protein in activated lymphocytes through the interaction with the NF-κB p65 subunit [217,218].
On the molecular level, genomic instability due to cellular stress is able to induce epigenetic changes, for example the release of HMGB1 (high mobility group box 1) proteins, which can function to exacerbate inflammatory responses [151]. On the cellular level, the accumulation of senescent and memory T-cells in immunological niches contributes to inflammatory processes which are then able to constitute an inflammatory state even at the systemic level [222]. This inflammation at the same time influences processes such as T-cell differentiation from naïve T lymphocytes.
Various studies over the last 15 years have demonstrated that many human diseases are inherently associated with inflammation and increased pro-inflammatory cytokines as well as short TL [5,239,240,241]. However, it is not entirely clear yet which factor is the cause and which the consequence or whether both scenarios are possible [242].
Diseases such as COPD (Chronic obstructive pulmonary disease) that are known to be associated with high inflammation also showed a high degree of telomere dysfunction and senescence in pulmonary vascular endothelial cells which was confirmed in telomerase knock-out mice where short telomeres at later generations result in higher levels of pro-inflammatory cytokines in proportion to telomere dysfunction in mouse lungs [239]. Another scenario was demonstrated by Jurk and colleagues who found that a chronic progressive inflammation in mice due to the knock-out of a NF-κB subunit induced telomere dysfunction, premature senescence as well as a much shorter lifespan [243]. The authors also described a decreased regeneration of organs such as liver and gut in this model which could, however, be ameliorated by an intervention with anti-inflammatory drugs such as ibuprofen as well as antioxidants. Importantly, the study found a feedback loop of NF-κB, COX-2 and ROS, which stabilized DNA damage in that system. The authors concluded that chronic systemic inflammation accelerated aging via a ROS-mediated exacerbation of telomere dysfunction and cell senescence without any external environmental factors.
In a human study on Japanese centenarians and semi-supercentenarians a better association of low inflammation levels with healthy aging was found than any association with longer TL and the inflammation level predicted all-cause mortality even better than chronological age [244]. Interestingly, the low inflammation score of these long-lived individuals seemed to be inherited since also their offspring showed that trait. In contrast, while both centenarians and their offspring maintained long TL, it was not a predictor for successful aging and longevity. This finding fits well with the suggestion about a possible advantage of a robust average TL compared to really long ones [230].
Inflammation can also influence TA, in particular in immune cells. Gizard and colleagues described that TA in human macrophages is induced by different inflammatory cytokines (TNFα, IL-1β) as well as LPS and oxidized LDL [202]. Importantly, the authors identified a novel NF-κB response element in the TERT promoter, where NF-κB is recruited to during inflammation and atherosclerotic stimulation, characterizing TERT as a direct NF-κB target gene [202].
There is a mutual regulation between the telomerase subunit TERT and the NF-κB, a master transcription factor for inflammation. While the hTERT promoter has a NF-κB binding site [202], hTERT can bind to the NF-κB subunit p65 to promote the transcription of NF-κB-dependent inflammatory genes such as TNFα and IL-6 [245,246]. However, these functions of TERT are independent of TA and telomeres. In contrast, an interferon-stimulated gene 15 (ISG15) can be upregulated by short telomeres [247] which could be an effect involving a telomere-position effect (TPE) while long telomeres are able to repress gene transcription over long distances (TPE-OLD) [248]. As part of the immune system’s stress response pathway, ISG15 can stimulate the pro-inflammatory IFNγ and might thereby link senescence-related inflammation with the aging process of the immune system [49]. In that way telomeres are able to influence multiple cellular functions and there might be further new functions to be discovered in the future.
Another type of a potential communication between telomeric sequences in the form of TERRA molecules in extracellular vesicles and inflammatory processes occurs in the innate immune system of the tissue microenvironment. While most TERRA molecules stay associated with their telomere of origin, some short TERRA molecules have been found in exosomes from the medium of cultivated lymphoid cells and named cell-free TERRA (cfTERRA) [249,250]. This specific TERRA form is around 200 nucleotides long and associated with histones [249]. The incubation of cfTERRA-containing exosomes with PBMCs induced the transcription of several inflammatory cytokine genes, including TNFα, IL6, and C-X-C chemokine 10 (CXCL10) [249]. Importantly, the authors found increased cfTERRA levels in exosomes when telomere dysfunction was experimentally triggered by expressing a dominant negative TRF2. Intriguingly, the exosomes from such damaged cells also possessed high levels of the DNA damage marker γH2AX as well as fragmented telomere repeat DNA.
Such cfTERRA containing exosomes have also been detected in human serum where they induce an inflammatory response via cytokines. Consequently, the authors emphasize that cfTERRA-containing exosomes display a telomere-associated molecular pattern (TAMP) and telomere-specific alarmin from dysfunctional telomeres as a novel form of inflammation induction in immune cells. They also suggest to employ cfTERRA as a potential biomarker for telomere-dysfunction and associated human diseases [250].
10. TL in Disease and Mortality
Telomere length is thought to be a relevant biomarker for aging and aging-related diseases. The most convincing correlation between compromised telomere maintenance/shortened telomeres and diseases comes from rare genetic diseases due to mutations in telomere-related genes, known as telomeropathies [102,251]. In these, due to mutations in telomerase compounds, TL already shortens during embryonic development leading to compromised tissue function preferentially in fast proliferating tissues such as bone marrow and skin. However, this large and rapid telomere loss cannot be compared to the much smaller, more gradual TS during adult life where individual differences in TL often overlay disease associations. One major disease-CVD seems to be an exception here since a good correlation has been described with TL [3,4,252,253,254].
Importantly, TL seems to predict biological age and thus survival and longevity and many epidemiological studies described an association of short TL with morbidity and mortality [3,255]. Other studies such as meta-analyses found a correlation between longer TL and many types of cancer as well as a correlation between short TL and degenerative diseases such as, for example, coronary heart disease, chronic kidney disease, rheumatoid arthritis, idiopathic pulmonary fibrosis, and also conditions such as facial aging [256].
However, most epidemiological studies are cross-sectional and thus cannot draw conclusions of causality because of huge inter-individual differences (see details above). In contrast, longitudinal studies are able to evaluate intra-individual rates of telomere shortening in study participants since they are able to consider differences in baseline TL.
One of the earliest studies to describe a clear association between TL in PBMCs and mortality on people aged 60-97 years was published in 2003 by Cawthon and co-authors [3]. The study was retrospective with a long follow-up period up to 15 years. It found that individuals with short telomeres had a nearly doubled risk of mortality during this period caused by a higher risk of infections and cardiovascular diseases while no such effect was found for other diseases such as cancer. Importantly, TL served as a predictor for death from diseases only between the ages of 60 and 74 while at higher ages there was only a very small correlation. This has been confirmed by other studies which demonstrated an absence of an association between TL and disease over the age of 85 in two large studies with individuals older than 85 years [257,258]. This could be due to a selection advantage and survival effect that occurs for people living to such a high age. Likewise, there seems to be no consistency between TLs as predictors for the risk of cancer. It has been found that both, long and short telomeres can be associated with risks for individual cancer types, however, there is now rather a consensus that longer LTL might be generally associated with a higher risk of cancer [164,256,259].
Another early cross-sectional study using LTL as predictors for disease outcomes was performed on a large age range of subjects from 18-98 years including a high number of geriatric patients [2]. As in other studies, a large variability in TL of around 2 kb was found for PBMCs between individuals of a given age, while there was a gradual decrease of average TL with increasing age. The average shortening rate was around 20 bp per year for PBMCs from healthy donors corresponding well to values found in other studies [187]. However, the main aim of the study was to analyze whether older patients who had suffered from a vascular stroke had different outcomes such as dementia depending on their TL in PBMCs. Indeed, patients who got demented after the stroke had significantly shorter telomeres (440 bp on average) in their PBMCs compared to aged-matched subjects or other patients who also had a stroke, but did not develop dementia afterwards and stayed cognitively competent. Based on that result the authors concluded that telomere length could be an independent predictor for the risk of vascular dementia after a stroke [2]. In addition, there were also skin biopsies taken from some of the participants and a good inter-individual correlation in TLs was found between cultured skin fibroblasts and PBMCs suggesting a strong genetic influence at the level of an individual as confirmed by others [192]. Other studies have confirmed the correlation of relative TL between different tissues from the same individual such as leukocytes and muscle [185,187,260] or glia cells and neurons [261]. The latter study described telomere shortening in glia cells, but not in neurons of adults while there was no difference in TL between these two cell types in infants confirming previous findings in other tissue types [185,187,260]. Importantly, the explanted skin fibroblasts from the above study [2] were additionally stressed by culturing them in normoxic and hyperoxic conditions together with some known fibroblast strains such as BJ, Wi38 and MRC-5. The group had shown previously that chronic hyperoxic stress accelerates telomere shortening rates (TSR) up to around 5-6 folds [39,77]. Additionally, the total antioxidant capacity (AOC) was examined in these cells to validate the hypothesis that the individual AOC might be responsible for stress sensitivity under increased oxidative stress such as hyperoxia. The study found a very good and statistically significant correlation between TSR and AOC including that of a high AOC in long-lived BJ fibroblasts and a low AOC in the shorter-lived MRC-5 and Wi38 strains while the patient-derived fibroblasts were in between these two extremes [2]. These results suggest that the antioxidant protection against increased oxidative stress might be a major determinant of TSR and telomere length in cultured telomerase-negative primary human fibroblasts. However, it is important to emphasize that telomeres in PBMCs and skin fibroblasts did not shorten due to the diseases such as acute ischemic stroke in brain vessels or dementia since tissues such as blood and skin seem rather not involved in those conditions. Instead, the authors draw the conclusion that the antioxidant make-up and thus the sensitivity of all tissues of an individual determines both the risk as well as the outcome after stroke to develop dementia and cognitive impairment because also their neurons would be less well protected against oxidative stress and tissue damage in the case of a low AOC [2]. It is well-known that both, ischemic and hemorrhagic strokes are caused by higher oxidative stress [262]. With other words, the individuals with short telomeres were most at risk to develop dementia after a stroke while their blood cell telomeres are not causally involved in the disease or its outcomes but rather be indicators of stress susceptibility. This might most likely also apply to many other conditions and diseases and is important to bear in mind for the interpretation of clinical study results.
However, the finding of an AOC-dependent TSR within an individual described above seems to be in contrast to findings of a similar TSR in 4 different tissues (leukocytes, skin, muscle and fat) in a cross-sectional study of adults of a similar age range (18-77 years) of around 24-26 bp/year independently of their replicative activity [187]. The authors conclude that differences in absolute TL between those tissues are based on genetic factors early during development while TSRs of those tissues seem to be similar during early life. However, study participants were all healthy subjects without increased oxidative stress due to disease conditions as described in [2].
The best way forward to find out more about individual TLs and TSRs in vivo are prospective longitudinal studies with large study cohorts or the use of biobanks to perform retrospective studies like the one from Cawthon and co-authors [3]. Moreover, animal studies, for example in birds, can be really helpful and indeed a correlation between long TL and low oxidative stress has been described in birds [263]. A recent longitudinal study in long-lived parrots confirmed the correlation between TL and high AOC/lower oxidative stress as well as longevity [264]. A similar correlation between low TSR and high life expectancy has been reported from other longevity studies in animals including birds previously [265,266,267]. While in disease-related studies the debate about cause and consequence for the correlation between TL and disease is still ongoing, for mortality there seems to be a clear causality regarding TL and underlying factors modulating them.
Various clinical epidemiological studies have found that TL can reflect the risk for chronic diseases such as CVD [268,269,270]. Interestingly, Xu and co-workers even found an inverse correlation between TL and CVD severity [269]. However, true mechanistic explanations are still missing whether short TL are really a cause for these degenerative diseases or just a consequence of disease-associated processes such as inflammation and increased oxidative stress. Possibly, both scenarios should be considered. Extensive longitudinal studies would be required to evaluate the telomere-life trajectory and to elucidate whether indeed subjects with short TL at birth, possibly together with examining the lifetime TSR, develop aging-related diseases earlier and have a shorter life expectancy.
Some human longitudinal studies have also found interesting sex-differences for the relation between TL and diseases such as mortality from CVD. For example, Epel and co-workers showed in a prospective longitudinal study for healthy participants between 70 and 79 years that in women the mortality risk from CVD was associated with basic TL while in men it was not the baseline TL but the age-dependent TSR determined over 2.5 years which predicted the risk for death from CVD in a follow-up period of 12 years [112]. However, as already mentioned above, there might be a selection effect at very high age. For example, there was no correlation found between TL and all-cause mortality in very old participants [257], while other studies described a good association between TL and self-reported health status [172,271] making it a potential biomarker for healthy aging.
However, the relationship between TL and diseases is not as simple as initially thought. Recently, studies that used a Mendelian randomization method to estimate a causality in cross-sectional epidemiological data, suggested that inherited short telomeres increase the risk of some degenerative diseases such as CVD and some neurodegenerative diseases, while long TL at baseline might rather predict a risk for cancer [259]. Similar findings were published by others who showed that diseases such as uterine fibroids, benign prostatic hyperplasia or several types of cancer such as sarcomas, are associated with long telomeres [272].
Importantly, several studies have established that the interindividual variation in LTL across newborns is as wide as in their parents or other adults. These findings support the important hypothesis that the correlation between measured LTLs in adults and various disease at later ages might already be largely pre-determined due to the high influence of inheritance for individual TLs [81].
11. Conclusions
The debate on how directly telomere biology affects life span is ongoing, but an association between telomere length and mortality as well as some age-associated diseases such as CVD has been demonstrated in many different studies already. In addition, telomere length has been suggested as a biological parameter that combines replicative history and exposure to environmental and oxidative stress [273] while others suggest that TL is rather causal to disease onset and development than being a mere biomarker [164].
Results from many studies indicate that TL regulation, in particular in white blood cells used in the vast majority of population-based studies, is a rather complex process that can be influenced by a multitude of internal and external factors and has a strong genetic component. Furthermore, due to different levels of TA in different subgroups of leukocytes as well as a changing ratio of different immune cell subgroups due to aging, disease and environmental influences, these parameters also need to be considered when using TL from blood cells. Consequently, results from cross-sectional population-based studies are often contradictory due to large genetically determined intra-individual differences in telomere length. Nevertheless, a good correlation of leukocyte TL with mortality in people until high age and with degenerative diseases such as CVD and atherosclerosis have been confirmed in several epidemiological studies as well as recent meta-analyses.
A strong inherited component of telomere length gets increasingly recognized from pioneering studies of A. Aviv’s group [81,185,186,187,188]. Importantly, such inherited differences in telomeres might already pre-dispose individuals to certain aging-related diseases [274]. In contrast, it is still disputed how much impact in these processes stems from individual telomere shortening rates. Telomeres are specifically vulnerable to inflicted DNA damage and thus might indicate a certain individual vulnerability to oxidative stress which can be from external (environmental toxins, lifestyle, nutrition, exercise etc. [275]) as well as internal processes such as mitochondrial dysfunction and inflammation [204,276] which all greatly contribute to increased oxidative stress (see summary Figure 6). Some studies emphasize that the vulnerability against oxidative stress and the set-up of antioxidant defense might also be genetically determined and characteristic for an individual/organism [2].
Figure 6 Influences on individual telomere length (TL) and telomere shortening rate (TSR). Inheritance mainly determines the initial TL at conception while other factors such as oxidative stress and genetic susceptibility to oxidative stress (for example via the antioxidant capacity) influence the different TSRs during adult life. In contrast, patients with telomeropathies (blue line) due to mutations in telomerase and telomere genes have very short TL at set-out reaching a critically short TL early in life and develop various degenerative diseases, predominantly in highly regenerative tissues such as the bone marrow.
Moreover, it gets increasingly recognized that in addition to evaluating TS in mitotic cells, also the accumulation of telomere damage in PM tissues is an important parameter that drives cellular senescence and organismal aging [30,141,277]. However, this important type of telomere dysfunction is very rarely analyzed in larger studies since this would require more invasive sampling techniques such as taking biopsies and also rather demanding analysis techniques that are not really suited for high-throughput analysis.
In general, the characterization of TL as a good biomarker for aging and related diseases is under constant discussion and it is now more and more accepted, that it might be still some indicator for these processes, but not as good as previously thought and should thus be rather combined with other biomarkers, for example epigenetic aging markers [182,278].
Finally, including new and recently discovered/recognized telomeric parameters such as extrachromosomal telomeric sequences [279,280] will improve telomere measurement methods, but might also make them more complex and possibly less suitable for clinical studies. In addition, a frequent lack of reproducibility between existing studies regarding telomere length measurements due to different study design, methods for TL determination, insufficient sample sizes as well as a high heterogeneity of study population poses additional challenges for the comparability of results [279].
Abbreviations
Author Contributions
The author did all the research work for this study.
Competing Interests
The author declares that no competing interests exist.
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