**3. Hallmarks of aging**

A completely different approach than to explain aging by theories is to describe it by its major characteristic features: "The Hallmarks of Aging" [32]:

#### **3.1. Genomic instability**

DNA is permanently exposed to a great variety of damage caused by ROS, lipid peroxidation products, environmental mutagens, hydrolytic reactions, UV irradiation and many more.

Besides chemical modifications to DNA, there can be single- and double-strand breaks, depurination as well as cross links between bases. Addition, deletion or substitution of bases is causing mutations. All together, these changes can also lead to epigenetic alterations causing changes in gene expression. To repair this damage efficiently is of utmost importance as can be seen in progeria or premature aging syndromes. In these diseases, defects in DNA repair systems are causing a dramatically accelerated aging process [33].

Usually, DNA damage is repaired very efficiently, however, the activity of the repair systems declines during aging [34].

#### **3.2. Telomere attrition**

Telomeres are protective caps on the ends of chromosomes preventing degradation or fusion of chromosome ends. At every cell division, telomeres are getting shorter and if they reach a critical length the cells, are not able to divide anymore. This telomere attrition eventually leads to replicative senescence. The degree of shortening is proportionate to risks of a number of aging diseases [35]. In particular, short telomeres lead to bone marrow failure causing anemia and immune senescence and to enterocolitis in the intestinal epithelium. Furthermore, short telomeres are causing premature onset of emphysema and pulmonary fibrosis in the lung, fibrosis in the liver and also osteoporosis [36]. In addition, short telomeres have an impact on gene expression through telomere position effects on nearby genes [37]. Through the expression of telomerase in mice, the normal aging process could be delayed [38]. In humans, 11 mutations inactivating a single gene are known which directly affect telomere maintenance and they lead to age-related diseases and accelerated aging [35]. Also a telomere biology disorder is the Hoyeraal-Hreidarsson (HH) syndrome which is caused by mutations in genes with telomeric functions. It is characterized by very short telomeres and affected individuals die in childhood mostly due to bone marrow failure [39]. Interestingly, it has been shown that in wild animals, telomeres shorten more slowly in slow-aging than in fast-aging ones [40].

## **3.3. Epigenetic alterations**

cause death of the cell eventually as is the case in Alzheimer patients. ROS are damaging cells in many locations in particular if they are produced excessively. On the other hand, ROS are important signaling molecules, therefore their complete removal by antioxidants is definitely counterproductive. In recent years, a modification of the free radical theory of aging gained attention: It claims that not ROS are driving the aging process but the dis-

Also a damage theory is the theory of "Inflamm-Aging" which claims that aging is caused by ongoing low level sterile inflammatory processes [28]. In fact all degenerative diseases in aging have an inflammatory component as there are: Alzheimer, Parkinson, arteriosclerosis, arthritis, multiple sclerosis, osteoporosis and diabetes type II [29]. Responsible for many inflammatory reactions are debris of dead cells and un-degradable protein aggregates which have not been removed completely. This was the basis to coin the term "Garb-Aging" (from garbage and aging) as an addition to inflamm-aging [30]. None of the so far mentioned theories, however, is able to explain all facets of the aging process. Therefore a new perspective was presented recently: "Aging: progressive decline in fitness due to the rising deleteriome" [31]. According to this theory neither the synthesis of biomolecules nor the repair systems in a cell are working absolute flawlessly. Furthermore there are chemical reactions between many biomolecule. The resulting compounds cannot be removed completely. That means that unwanted reaction products as well as non-repaired damage (= deleteriome) increases

A completely different approach than to explain aging by theories is to describe it by its major

DNA is permanently exposed to a great variety of damage caused by ROS, lipid peroxidation products, environmental mutagens, hydrolytic reactions, UV irradiation and many more.

Besides chemical modifications to DNA, there can be single- and double-strand breaks, depurination as well as cross links between bases. Addition, deletion or substitution of bases is causing mutations. All together, these changes can also lead to epigenetic alterations causing changes in gene expression. To repair this damage efficiently is of utmost importance as can be seen in progeria or premature aging syndromes. In these diseases, defects in DNA repair

Usually, DNA damage is repaired very efficiently, however, the activity of the repair systems

Telomeres are protective caps on the ends of chromosomes preventing degradation or fusion of chromosome ends. At every cell division, telomeres are getting shorter and if they reach a

turbed redox homeostasis is a major culprit [26, 27].

with time and causes the aging process.

characteristic features: "The Hallmarks of Aging" [32]:

systems are causing a dramatically accelerated aging process [33].

**3. Hallmarks of aging**

118 Gerontology

**3.1. Genomic instability**

declines during aging [34].

**3.2. Telomere attrition**

Epigenetic changes are comprising alterations in histone marks, DNA methylation, nucleosome positioning and non-coding RNAs [41]. Histone modifications and methylation patterns of CpG islands have a tremendous influence on gene expression. For a set of 353 CpG islands, a clear correlation with age could be demonstrated. Of these, 193 CpGs get hypermethylated and 160 get hypomethylated during aging [42]. According to its reliable changes in methylation status, this set has been termed the epigenetic clock [27, 42, 43]. With this epigenetic clock, it is possible to predict the biological age and an age-related functional decline. Furthermore, it has been demonstrated that lifestyle factors like diet, exercise and education have an influence on this epigenetic clock [44]. In addition to DNA, the histones are subject to modifications (acetylation, methylation, phosphorylation and more). There are also a number of methylation marks that are changing with age. But the present picture is less clear than with DNA methylation [41]. A lot of attention has been focused on sirtuins. Sirtuins are class III histone deacetylases, which need NAD<sup>+</sup> as a cofactor and remove acetyl groups from previously modified lysines in the histone N-terminal tails. By removing the acetyl groups, lysines regain their positive charge and bind more tightly to DNA. The result is a more compact chromatin structure and down-regulation of gene expression. The removal of histone acetyl groups by sirtuins results in an extension of lifespan [45]. In total, seven sirtuins are known, of which SIRT1, SIRT6 and SIRT7 are localized in the nucleus. SIRT2 is predominantly found in the cytoplasm and is only localized to chromatin during the G/M phase of the cell cycle. SIRT3, SIRT4 and SIRT5 are the three mitochondrial deactelyases. Sirtuins do not only deacetylate histones but regulate the activity of a number of other proteins too. This way they play a central role in regulatory networks important for aging and longevity [46]. Mutant mice where single sirtuin genes have been deleted show a number of different pathologies connected to metabolism, cancer and inflammation [47].

#### **3.4. Loss of proteostasis**

Proteins not only have to be synthesized but they have to be removed and degraded eventually. Among many others, there are two major ways to remove damaged proteins: either to degrade them by the proteasome or via autophagy [48, 49]. In addition, the cell has chaperones. These proteins help to fold proteins correctly or enable the renaturation of already denatured proteins. If refolding is impossible, chaperons are also able to target misfolded proteins to the proteasome. Therefore proteostasis, the maintenance of an intact proteome, includes not only synthesis and degradation of proteins but also folding and conformational maintenance. The disturbance of proteostasis is considered to be a major cause of aging [50, 51]. Not only the amount of chaperones is decreasing [52], but also the proteasomal activity and autophagy are declining during aging. This decline causes an accumulation of denatured proteins which have the tendency to form aggregates that cannot be removed by the cell anymore. These aggregates are detrimental to the cell and can even cause death of the cell (e.g. nerve cells in Alzheimer and Parkinson patients) [53]. Furthermore, it has been demonstrated that long-living animals have less denatured proteins than short-living ones [54]. In addition, the activity of the proteasome is remarkably higher in the long-living naked mole rat than in the short-lived mouse [55]. Particularly interesting in this respect is that experimental interventions which reduce the aging process are stimulating autophagy like caloric restriction, rapamycin, metformin, resveratrol and spermidine [56].

oxygen to water. If accidentially oxygen gets only one electron, it leads to the production of ROS instead of water. This has inspired Harman already in 1956 to present his "Free Radical Theory of Aging," which he has repeatedly improved [18]. Furthermore, it has been demonstrated that ROS are not only causing damage but also are important signaling molecules, which are able to

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The events of biogenesis, fusion, fission and mitophagy are collected under the term mitochondrial dynamics [65]. Function as well as quality of mitochondria is regulated by mitochondrial dynamics. Nutrients in excess cause fragmentation (fission) of mitochondria and a low level of nutrients leads to fusion and elongation [66]. Mitochondrial dynamics is also influenced by external signals like hormones, nutrients and physical exercise. A number of pathways are involved like mTOR, AMP-activated kinase and sirtuins [67]. In particular, sirtuins play an important role as they do not regulate a few target enzymes but regulate functional clusters (e.g. TCA cycle, fatty acid metabolism, electron transport chain and others). This way they are involved in the regulation of ROS-mediated signaling pathways as well as in the detoxification of damaging ROS. Furthermore they regulate metabolic plasticity. SIRT3, for example, promotes switching to fatty acid oxidation upon caloric restriction [68]. The modulation of metabolic changes plays a crucial role in senescence too. In addition, defect mitochondria are stimulating inflammatory reactions which are triggering inflamm-aging [69]. Altogether, mitochondrial dysfunction leads to a number of age-related diseases including metabolic, cardiovascular and neurodegenerative pathologies, sarcopenia and fibrosis in

All these facts demonstrate that the quality of mitochondria has a tremendous impact on the

Cellular senescence is characterized by an irreversible arrest of the cell cycle. This means that cellular senescence can only affect cells that are able to divide like stem cells, progenitor cells or cells that are not yet terminally differentiated. This phenomenon has been discovered with cells in cell culture. It turned out that they cannot divide without limits but stop growing after

The irreversible cell cycle arrest can be induced by erosion of telomeres, substantial DNA damage, oxidative stress, overexpression of oncogenes, mitochondrial dysfunction and proteotoxicity [73]. Cells in the state of senescence change their morphology, they are getting larger and there are massive changes in the organization of chromatin [74]. Furthermore, they start to secret a large number of proteins. The sum of all these proteins is called the senescence-associated secretory phenotype (SASP). Among these proteins are pro-inflammatory cytokines, chemokines, growth factors and matrix metalloproteases (MMPs). The pro-inflammatory cytokines are causing local sterile inflammations which contribute substantially to "inflammaging". They attract cells of the immune system which are killing senescent cells. The overproportional increase of senescent cells during aging is probably due to a decline in immune function [75]. An important additional feature of senescent cells is the active suppression of apoptosis [76]. At the level of gene expression, permanent cell cycle arrest is mediated by the protein p16Ink4a which is an inhibitor of the cyclin dependent kinases 4 and 6 (CDK4 and CDK6).

regulate many pathways. For example, ROS can induce autophagy (mitophagy) [64].

different organs [65].

aging process [70, 71].

**3.7. Cellular senescence**

about 40–50 cell divisions [72].

#### **3.5. Deregulated nutrient sensing**

Nutrient sensing is of utmost importance for every cell. The major nutrient sensing pathways that are also longevity pathways are [57]:


IGF-1 is like insulin a growth factor for many cells and acts via the insulin and IGF-1 signaling (IIS) pathway. The down-regulation of this pathway leads to a prolonged lifespan [58]. IGF-1 but also EGF and high amino acid levels are activating the mTOR pathway which stimulates protein synthesis and growth in general but down-regulates autophagy [59]. AMPK is the sensor and regulator for energy metabolism and homeostasis of the cell. AMPK activity can extend the lifespan of yeast, *C. elegans* and drosophila and the healthspan of mice [60].

NAD-dependent sirtuins are a family of deacylases which not only deacetylate histones but modify a large number of non-histone proteins too. They show impressive activities to prevent diseases and some aspects of aging [61].

During aging, the synthesis of these sensor proteins is reduced however [62].

The different signal transduction pathways that are sensing the availability of nutrients are deregulated during aging by metabolic diseases [63].

#### **3.6. Mitochondrial dysfunction**

As mitochondria are not only the power plants of the cell but also important signaling centers they play a central role in the aging process. For energy production in form of ATP, they reduce oxygen to water. If accidentially oxygen gets only one electron, it leads to the production of ROS instead of water. This has inspired Harman already in 1956 to present his "Free Radical Theory of Aging," which he has repeatedly improved [18]. Furthermore, it has been demonstrated that ROS are not only causing damage but also are important signaling molecules, which are able to regulate many pathways. For example, ROS can induce autophagy (mitophagy) [64].

The events of biogenesis, fusion, fission and mitophagy are collected under the term mitochondrial dynamics [65]. Function as well as quality of mitochondria is regulated by mitochondrial dynamics. Nutrients in excess cause fragmentation (fission) of mitochondria and a low level of nutrients leads to fusion and elongation [66]. Mitochondrial dynamics is also influenced by external signals like hormones, nutrients and physical exercise. A number of pathways are involved like mTOR, AMP-activated kinase and sirtuins [67]. In particular, sirtuins play an important role as they do not regulate a few target enzymes but regulate functional clusters (e.g. TCA cycle, fatty acid metabolism, electron transport chain and others). This way they are involved in the regulation of ROS-mediated signaling pathways as well as in the detoxification of damaging ROS. Furthermore they regulate metabolic plasticity. SIRT3, for example, promotes switching to fatty acid oxidation upon caloric restriction [68]. The modulation of metabolic changes plays a crucial role in senescence too. In addition, defect mitochondria are stimulating inflammatory reactions which are triggering inflamm-aging [69]. Altogether, mitochondrial dysfunction leads to a number of age-related diseases including metabolic, cardiovascular and neurodegenerative pathologies, sarcopenia and fibrosis in different organs [65].

All these facts demonstrate that the quality of mitochondria has a tremendous impact on the aging process [70, 71].

### **3.7. Cellular senescence**

them by the proteasome or via autophagy [48, 49]. In addition, the cell has chaperones. These proteins help to fold proteins correctly or enable the renaturation of already denatured proteins. If refolding is impossible, chaperons are also able to target misfolded proteins to the proteasome. Therefore proteostasis, the maintenance of an intact proteome, includes not only synthesis and degradation of proteins but also folding and conformational maintenance. The disturbance of proteostasis is considered to be a major cause of aging [50, 51]. Not only the amount of chaperones is decreasing [52], but also the proteasomal activity and autophagy are declining during aging. This decline causes an accumulation of denatured proteins which have the tendency to form aggregates that cannot be removed by the cell anymore. These aggregates are detrimental to the cell and can even cause death of the cell (e.g. nerve cells in Alzheimer and Parkinson patients) [53]. Furthermore, it has been demonstrated that long-living animals have less denatured proteins than short-living ones [54]. In addition, the activity of the proteasome is remarkably higher in the long-living naked mole rat than in the short-lived mouse [55]. Particularly interesting in this respect is that experimental interventions which reduce the aging process are stimulating

autophagy like caloric restriction, rapamycin, metformin, resveratrol and spermidine [56].

Nutrient sensing is of utmost importance for every cell. The major nutrient sensing pathways

IGF-1 is like insulin a growth factor for many cells and acts via the insulin and IGF-1 signaling (IIS) pathway. The down-regulation of this pathway leads to a prolonged lifespan [58]. IGF-1 but also EGF and high amino acid levels are activating the mTOR pathway which stimulates protein synthesis and growth in general but down-regulates autophagy [59]. AMPK is the sensor and regulator for energy metabolism and homeostasis of the cell. AMPK activity can

NAD-dependent sirtuins are a family of deacylases which not only deacetylate histones but modify a large number of non-histone proteins too. They show impressive activities to pre-

The different signal transduction pathways that are sensing the availability of nutrients are

As mitochondria are not only the power plants of the cell but also important signaling centers they play a central role in the aging process. For energy production in form of ATP, they reduce

extend the lifespan of yeast, *C. elegans* and drosophila and the healthspan of mice [60].

During aging, the synthesis of these sensor proteins is reduced however [62].

**3.5. Deregulated nutrient sensing**

that are also longevity pathways are [57]:

• IGF-1 and insulin signaling pathway

dependent sirtuins

• AMP-activated protein kinase (AMPK) pathway

vent diseases and some aspects of aging [61].

**3.6. Mitochondrial dysfunction**

deregulated during aging by metabolic diseases [63].

• mTOR pathway

• NAD<sup>+</sup>

120 Gerontology

Cellular senescence is characterized by an irreversible arrest of the cell cycle. This means that cellular senescence can only affect cells that are able to divide like stem cells, progenitor cells or cells that are not yet terminally differentiated. This phenomenon has been discovered with cells in cell culture. It turned out that they cannot divide without limits but stop growing after about 40–50 cell divisions [72].

The irreversible cell cycle arrest can be induced by erosion of telomeres, substantial DNA damage, oxidative stress, overexpression of oncogenes, mitochondrial dysfunction and proteotoxicity [73]. Cells in the state of senescence change their morphology, they are getting larger and there are massive changes in the organization of chromatin [74]. Furthermore, they start to secret a large number of proteins. The sum of all these proteins is called the senescence-associated secretory phenotype (SASP). Among these proteins are pro-inflammatory cytokines, chemokines, growth factors and matrix metalloproteases (MMPs). The pro-inflammatory cytokines are causing local sterile inflammations which contribute substantially to "inflammaging". They attract cells of the immune system which are killing senescent cells. The overproportional increase of senescent cells during aging is probably due to a decline in immune function [75]. An important additional feature of senescent cells is the active suppression of apoptosis [76]. At the level of gene expression, permanent cell cycle arrest is mediated by the protein p16Ink4a which is an inhibitor of the cyclin dependent kinases 4 and 6 (CDK4 and CDK6). In healthy young cells, p16Ink4a expression is low or undetectable but increases dramatically in senescent cells [9]. This way it is evident that an essential function of senescence (maybe the most important one for the organism) is to pull the emergency brake to prevent uncontrolled cell division which otherwise could cause tumor formation. Senescent cells have also an important additional function in wound healing. After a wound has been inflicted many cells are produced in excess to close the wound. During the subsequent remodeling process, the surplus of cells is entering senescence and will be removed by the immune system. For years it has been discussed by researchers if cellular senescence has any influence on the aging process itself. During the past few years, scientists came to the conclusion that cellular senescence is one of the major causes of aging [77]. In genetically modified mice, it was already possible to delete senescent cells (p16Ink4a positive cells). These animals showed less age-related pathologies, an improved healthspan and a prolonged median lifespan [9, 11]. Therefore, there are already a number of different interventions under investigation how senescent cells can be removed from the human body [78, 79]. To succeed in this respect could dramatically improve human healthspan. Senescent cells are detrimental to the function of organs they are residing in and this way they have a tremendous impact on age-dependent degenerative diseases [12, 78].

cells available for the regeneration of muscle tissue [87]. In a similar way, the prolonged signaling of the growth hormone (GH)/insulin/insulin-like growth factors (IGF) axis is considered to cause a depletion of stem cells [88]. There are also areas in the adult brain where stem cells are residing: in the dentate gyrus of the hippocampus in the hypothalamus and in the subventricular zone of the lateral ventricles [89]. Like in other tissues, there are age-related changes in the stem cell niche and the number of neural stem cells is declining during aging. Not only the numbers of stem cells are decreasing during aging but also the proliferation of the developing precursor cells will be damped via an elevated concentration of TGF-beta1. This way the production of new neurons is reduced while the generation of oligodendroglia remains at about the same level [90]. Furthermore, sterile micro-inflammation in the hypothalamus can cause a reduction of neural stem cells which in turn leads to a reduction of cognitive functions [91]. In addition to the number of stem cells and the contribution of the stem cell niche, the regenerative capacity will be influenced by systemic factors. Via parabiosis experiments (connecting an old mouse to a young one), it was possible to correct malfunction of old satellite cells in muscle tissue. These satellite cells could be reactivated again. In a similar way, it was possible to improve the function of stem cells and neurogenesis in old brains. The proteins responsible for this activity could be identified as growth differentiation factor 11 (GDF11) and oxytocin [92]. There are numerous factors that regulate the biological function of stem cells. Presently, it seems that the most important ones are metabolism and epigenetic changes [80]. As excessive nutrient sensing leads to premature depletion of adult neural stem cells [89] and chronic activation of mTOR leads to loss of stem cells in the airway epithelium of the mouse [93].

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The regenerative capacity of stem cells is independently influenced by intrinsic and extrinsic determinants [92]. An intrinsic determinant is the maintenance of autophagy. A failure of autophagy in old satellite cells leads to senescence eventually [94]. Among the extrinsic factors, growth hormone/insulin/IGF-1 (somatotrophic axis) is a center piece for the regulation of growth in the mammalian organism. Mouse mutants with defects in the biosynthesis of the growth hormone (Ames dwarf mice, Snell dwarf mice or GHRKO-mice (GH receptor deletion)) are considerably smaller than wild type mice but have an approximately 50% longer lifespan [95]. In humans, the amount of growth hormone and IGF-1 in the circulation is changing during aging. The highest level is reached during the second decade where growth is most prominent. Afterwards the concentration is going down continuously until it reaches a low plateau during the sixth decade. Humans with genetic polymorphisms resulting in a reduced activity of IGF-1 show a significantly increased lifespan. An elevated concentration of IGF-1 is correlated to a higher risk for some tumors [96]. There is also an altered communication between muscle stem cells and the environment. Growth hormone is important for the maintenance of muscle mass [97]. IGF-1 is modulating the differentiation of muscle progenitor cells (myoblasts) and influencing satellite cells [98]. An increased aging of muscle stem cells is caused by an elevated concentration of Wnt-proteins (e.g. Wnt3A) [99]. As an antagonist of Wnt/β-catenin signaling acts the protein klotho. Unfortunately, the amount of klotho in the circulation is decreasing during aging. In the mouse, the silencing of the klotho gene triggered a rapid aging process [100]. Klotho is essential for the homeostasis of mineral metabolism (in particular phosphate) but it also modulates the signaling pathways of IGF-1 and Wnt. The deletion of the klotho gene in mice reduces their lifespan to 2–3 months which

**3.9. Altered intercellular communication**

#### **3.8. Stem cell exhaustion**

Stem cells are of utmost importance for tissue homeostasis and regeneration and stem cell exhaustion is among the most significant hallmarks of aging. Stem cell exhaustion is leading to a reduced regenerative capacity during the aging process. Premature stem cell exhaustion is also seen in age-related diseases [80]. Stem cells are usually very small remaining in a state of quiescence. This state is characterized by low metabolism and the presence of few mitochondria. From dormancy, they can be activated to replace lost stem cells or to produce transit amplifying cells which will provide many cells for repair or regeneration of their particular tissue. During this differentiation process, they are going through a developmental program which is tuning them precisely to their new function [81].

There are tissues with a very high turnover of cells like bone marrow, intestine and the epidermis of the skin. There are also tissues where stem cells get activated rarely like muscle and brain. Essential for survival and quiescence of stem cells is their immediate environment which is defined as the stem cell niche. The stem cell niche comprises proteins of the extracellular matrix and surrounding cells which secrete a number of growth regulating proteins (Wnts, BMPs, EGF and Notch). It is essentially regulating the state of quiescence [82, 83]. Different drivers of aging (telomere attrition, cellular senescence, DNA damage, epigenetic alterations, nutrient sensing and disturbed proteostasis) have their impact on stem cells too and are responsible for stem cell aging [84]. As stem cells usually stay in the state of quiescence and divide rarely, many pro-aging impacts affect the stem cells via their niche. Muscle stem cells, so called satellite cells, rarely divide, but proliferate massively upon demand. They produce myoblasts which are the precursor cells necessary for the regeneration process of the muscle. If old satellite cells are transplanted into young muscle tissue their regenerative capacity increases which demonstrates the influence of the young niche [85, 86]. The opposite is true for transforming growth factor beta (TGF-beta1). This factor is produced by the niche and reduces the proliferative potential of satellite cells. During aging under certain circumstances the niche increases the production of fibroblast growth factor 2 (FGF2). This triggers the stem cells to leave quiescence and start to divide which eventually leads to a reduction of satellite cells available for the regeneration of muscle tissue [87]. In a similar way, the prolonged signaling of the growth hormone (GH)/insulin/insulin-like growth factors (IGF) axis is considered to cause a depletion of stem cells [88]. There are also areas in the adult brain where stem cells are residing: in the dentate gyrus of the hippocampus in the hypothalamus and in the subventricular zone of the lateral ventricles [89]. Like in other tissues, there are age-related changes in the stem cell niche and the number of neural stem cells is declining during aging. Not only the numbers of stem cells are decreasing during aging but also the proliferation of the developing precursor cells will be damped via an elevated concentration of TGF-beta1. This way the production of new neurons is reduced while the generation of oligodendroglia remains at about the same level [90]. Furthermore, sterile micro-inflammation in the hypothalamus can cause a reduction of neural stem cells which in turn leads to a reduction of cognitive functions [91]. In addition to the number of stem cells and the contribution of the stem cell niche, the regenerative capacity will be influenced by systemic factors. Via parabiosis experiments (connecting an old mouse to a young one), it was possible to correct malfunction of old satellite cells in muscle tissue. These satellite cells could be reactivated again. In a similar way, it was possible to improve the function of stem cells and neurogenesis in old brains. The proteins responsible for this activity could be identified as growth differentiation factor 11 (GDF11) and oxytocin [92]. There are numerous factors that regulate the biological function of stem cells. Presently, it seems that the most important ones are metabolism and epigenetic changes [80]. As excessive nutrient sensing leads to premature depletion of adult neural stem cells [89] and chronic activation of mTOR leads to loss of stem cells in the airway epithelium of the mouse [93].

#### **3.9. Altered intercellular communication**

In healthy young cells, p16Ink4a expression is low or undetectable but increases dramatically in senescent cells [9]. This way it is evident that an essential function of senescence (maybe the most important one for the organism) is to pull the emergency brake to prevent uncontrolled cell division which otherwise could cause tumor formation. Senescent cells have also an important additional function in wound healing. After a wound has been inflicted many cells are produced in excess to close the wound. During the subsequent remodeling process, the surplus of cells is entering senescence and will be removed by the immune system. For years it has been discussed by researchers if cellular senescence has any influence on the aging process itself. During the past few years, scientists came to the conclusion that cellular senescence is one of the major causes of aging [77]. In genetically modified mice, it was already possible to delete senescent cells (p16Ink4a positive cells). These animals showed less age-related pathologies, an improved healthspan and a prolonged median lifespan [9, 11]. Therefore, there are already a number of different interventions under investigation how senescent cells can be removed from the human body [78, 79]. To succeed in this respect could dramatically improve human healthspan. Senescent cells are detrimental to the function of organs they are residing in and this way they have a tremendous impact on age-dependent degenerative diseases [12, 78].

Stem cells are of utmost importance for tissue homeostasis and regeneration and stem cell exhaustion is among the most significant hallmarks of aging. Stem cell exhaustion is leading to a reduced regenerative capacity during the aging process. Premature stem cell exhaustion is also seen in age-related diseases [80]. Stem cells are usually very small remaining in a state of quiescence. This state is characterized by low metabolism and the presence of few mitochondria. From dormancy, they can be activated to replace lost stem cells or to produce transit amplifying cells which will provide many cells for repair or regeneration of their particular tissue. During this differentiation process, they are going through a developmental program

There are tissues with a very high turnover of cells like bone marrow, intestine and the epidermis of the skin. There are also tissues where stem cells get activated rarely like muscle and brain. Essential for survival and quiescence of stem cells is their immediate environment which is defined as the stem cell niche. The stem cell niche comprises proteins of the extracellular matrix and surrounding cells which secrete a number of growth regulating proteins (Wnts, BMPs, EGF and Notch). It is essentially regulating the state of quiescence [82, 83]. Different drivers of aging (telomere attrition, cellular senescence, DNA damage, epigenetic alterations, nutrient sensing and disturbed proteostasis) have their impact on stem cells too and are responsible for stem cell aging [84]. As stem cells usually stay in the state of quiescence and divide rarely, many pro-aging impacts affect the stem cells via their niche. Muscle stem cells, so called satellite cells, rarely divide, but proliferate massively upon demand. They produce myoblasts which are the precursor cells necessary for the regeneration process of the muscle. If old satellite cells are transplanted into young muscle tissue their regenerative capacity increases which demonstrates the influence of the young niche [85, 86]. The opposite is true for transforming growth factor beta (TGF-beta1). This factor is produced by the niche and reduces the proliferative potential of satellite cells. During aging under certain circumstances the niche increases the production of fibroblast growth factor 2 (FGF2). This triggers the stem cells to leave quiescence and start to divide which eventually leads to a reduction of satellite

**3.8. Stem cell exhaustion**

122 Gerontology

which is tuning them precisely to their new function [81].

The regenerative capacity of stem cells is independently influenced by intrinsic and extrinsic determinants [92]. An intrinsic determinant is the maintenance of autophagy. A failure of autophagy in old satellite cells leads to senescence eventually [94]. Among the extrinsic factors, growth hormone/insulin/IGF-1 (somatotrophic axis) is a center piece for the regulation of growth in the mammalian organism. Mouse mutants with defects in the biosynthesis of the growth hormone (Ames dwarf mice, Snell dwarf mice or GHRKO-mice (GH receptor deletion)) are considerably smaller than wild type mice but have an approximately 50% longer lifespan [95]. In humans, the amount of growth hormone and IGF-1 in the circulation is changing during aging. The highest level is reached during the second decade where growth is most prominent. Afterwards the concentration is going down continuously until it reaches a low plateau during the sixth decade. Humans with genetic polymorphisms resulting in a reduced activity of IGF-1 show a significantly increased lifespan. An elevated concentration of IGF-1 is correlated to a higher risk for some tumors [96]. There is also an altered communication between muscle stem cells and the environment. Growth hormone is important for the maintenance of muscle mass [97]. IGF-1 is modulating the differentiation of muscle progenitor cells (myoblasts) and influencing satellite cells [98]. An increased aging of muscle stem cells is caused by an elevated concentration of Wnt-proteins (e.g. Wnt3A) [99]. As an antagonist of Wnt/β-catenin signaling acts the protein klotho. Unfortunately, the amount of klotho in the circulation is decreasing during aging. In the mouse, the silencing of the klotho gene triggered a rapid aging process [100]. Klotho is essential for the homeostasis of mineral metabolism (in particular phosphate) but it also modulates the signaling pathways of IGF-1 and Wnt. The deletion of the klotho gene in mice reduces their lifespan to 2–3 months which is only about 10% of their regular lifespan [101]. A remarkable activity has also been demonstrated for GDF11 which improves regeneration in old organisms and serum levels of GDF11 are significantly lower in old individuals [102]. An increased regenerative activity has been shown for bone [103], brain [104], skeletal muscle [105] and heart [106].

maximum oxygen consumption and reduced levels of cholesterol and triglycerides in the blood, but it also improves physical and psychical conditions in old age [126]. Although a number of physiological parameters can be improved considerably by physical training, the protective function for the cardiovascular system are about twice as high as can be explained by these parameters only. Therefore there are still many open questions concerning the molecular mechanisms which are activated by physical training [127]. Very well documented is, however, the positive effect on the brain and in particular on cognitive functions and the stimulation of neuronal growth in the hippocampus, an area critically important for memory processes [128]. Physical exercise increases hippocampal volume, functional connectivity and improved connectivity between the default mode network and the prefrontal cortex [129]. In this context, it should also be mentioned that physical exercise leads to a significant improve-

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Already in 1935 it has been demonstrated on rats that reducing the amount of food intake can extend the lifespan by 30% [131]. This experiment has been repeated many times and it turned out that animals are not only living longer but they also show less age-related deficits. During the past years, it has been demonstrated that the amount of calories is less important than the amount of proteins. Therefore the term caloric restriction has been replaced in most cases by the term dietary restriction. In addition to the amount of food, the timing of food uptake is important. Animals getting their food evenly distributed during the day did not show positive effects but animals fed only once a day did show the positive effects. Also did fasting every second day result in an increase of lifespan by 30% [132]. Altogether a great many experiments have been performed concerning this topic and results are sometimes contradictory. Some authors are pointing out explicitly that it is necessary to test many different combinations of carbohydrates and proteins in a single experiment. It has been demonstrated that a relation of 1:10 (proteins:carbohydrates) results in the longest lifespan in mice. Remarkable in this respect is the fact that the traditional diet of the population of Okinawa consists of protein to carbohydrates in a relation of 9:85 and it is well documented that the people of Okinawa have the highest life expectancy worldwide [133]. It has to be mentioned that not only permanent dietary restriction is effective but intermittent fasting too. In rats and mice as well as in humans, there are profound health benefits. Results of intermittent fasting (2 days per week or every other day) decreased insulin levels, increased resistance to stress of heart and brain, reduced inflammation, enhanced autophagy, mitochondrial health and DNA repair [134]. Concerning DNA repair, the following experiment is really remarkable: mice lacking the DNA excision repair gene Ercc1 are aging very fast with a lifespan of 4–6 months. If they are subjected to a dietary restriction of 30%, this treatment triples their lifespan [33]. The single cell senses the availability of nutrition via nutrient sensing pathways which are GH/insulin/IGF-1, mTOR, sirtuins and AMPK and via these pathways the metabolic influence on the aging process is regulated [57].

Attenuating the signaling of the somatotrophic axis results in an increased lifespan. This has been demonstrated in animal models, in genetic polymorphisms or functional mutations in

ment of memory functions in Alzheimer patients [130].

**4.2. Caloric restriction/dietary restriction**

*4.2.1. The somatotrophic axis (GH/insulin/IGF-1)*

Furthermore, a number of chemokines (CCL2, CCL11, CCL12 and CCL19) have been identified via parabiosis experiments and they have been correlated with impaired neurogenesis in old individuals [107]. Other potential pro-aging factors that increase during lifetime are TGF-beta1, IL-6 and TNF-alpha [107]. Beta2microglobulin too is a systemic pro-aging factor triggering age-related cognitive impairment [108].

Another pro-aging factor is the plasminogen activator inhibitor 1 (PAI-1) which is secreted by senescent cells. It induces the accumulation of p16Ink4a leading to cellular senescence [109]. An anti-aging factor is kallistatin which inhibits oxidative stress and inflammation. It is also able to down-regulate the miRNA synthesis of miR21 and miR-34a, thereby reducing vascular senescence and aging [110]. The protein tissue inhibitor of metalloproteinase 2 (TIMP 2) was isolated from human umbilical cord. It is an anti-aging protein which revitalizes the hippocampus, increases synaptic plasticity and improves cognitive function [111]. The intercellular communication is also altered by numerous pro-inflammatory cytokines which are released by senescent cells. These cytokines are causing inflammatory processes [112]. Furthermore, inflammasomes in the cells of the innate immune system can be activated by DAMPs (damage-associated molecular patterns) [113]. DAMPS are comprised of debris of necrotic cells, amyloide fibers, HMGB1, heat shock proteins, crystals of cholesterol and uric acid. Activated inflammasomes are causing the release of interleukins IL-1beta and IL-18 [114]. These interleukins trigger inflammatory reactions in the surrounding tissue which are causing agerelated diseases [115], among them Alzheimer´s disease [116].

Exosomes provide an additional possibility for intercellular communication. They are small lipid vesicles which are secreted by the cell and they carry proteins and functional RNAs [117]. They can contact nearby cells or they can be distributed via the circulation across the whole organism. They help the cell to get rid of toxic protein waste [118] or to contribute to intercellular communication [119]. In the latter case, predominantly miRNAs play an important function [120]. During aging, the amount of exosomes in the blood stay more or less constant. Their content, however, becomes more pro-inflammatory [121]. Recently, it has been shown that they also play a role in senescence and aging [122].
