Chapter 2. : Aging at the Molecular and Cellular Level

 What are the hallmarks of aging at the molecular and cellular level?

Long quote:

"This Review enumerates nine tentative hallmarks that represent common denominators of aging in different organisms, with special emphasis on mammalian aging. These hallmarks are: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. A major challenge is to dissect the interconnectedness between the candidate hallmarks and their relative contributions to aging, with the final goal of identifying pharmaceutical targets to improve human health during aging, with minimal side effects." 1.
In summary of the aforementioned, the 9 hallmarks of aging at the molecular and cellular level are: 1. Stem Cell Exhaustion; 2. Altered inter-cellular communication; 3. Genomic Instability; 4. Telomere attrition; 5. Epigenetic alterations; 6. Loss of proteostasis; 7. Deregulated nutrient sensing; 8. Mitochondrial dysfunction and,; 9. Cellular Senescence.  2.

In order to further define the aforementioned hallmarks, a definition of the aforementioned follows.
 
 1. Stem Cell Exhaustion

Long Quote:

"The stem cell theory of aging postulates that the aging process is the result of the inability of various types of stem cells to continue to replenish the tissues of an organism with functional differentiated cells capable of maintaining that tissue's (or organ's) original function. Damage and error accumulation in genetic material is always a problem for systems regardless of the age. The number of stem cells in young people is very much higher than older people and this cause a better and more efficient replacement mechanism in the young contrary to the old. In other words, aging is not a matter of the increase of damage, but a matter of failure to replace it due to decreased number of stem cells. Stem cells decrease in number and tend to lose the ability to differentiate into progenies or lymphoid lineages and myeloid lineages.
Maintaining the dynamic balance of stem cell pools requires several conditions. Balancing proliferation and quiescence along with homing (See niche) and self-renewal of hematopoietic stem cells are favoring elements of stem cell pool maintenance while differentiation, mobilization and senescence are detrimental elements. These detrimental effects will eventually cause apoptosis." 3.
 
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"As we age, our stem cells eventually lose their ability to divide. Furthermore, we are unable to replace the stem cells that have migrated, differentiated, or died. As a result, we show outward symbols of aging, such as grey hair. While the decrease in the renewal of stem cells certainly leads to age-related disorders, it is clear that this “stem cell exhaustion” is really a consequence of DNA damage, deregulated nutrient sensing, senescence, and other processes already mentioned—in other words, it might be argued that it is not a “true” hallmark. Nevertheless, because of their unique role in determining cell fate in a tissue-specific way, stem cells can reveal ways that tissues interact during the aging of a complex organism and possibly redirect the fate of aging tissues upon transplantation." 4.

What is Apoptosis?

Long Quote:

"Apoptosis (from Ancient Greek ἀπόπτωσις "falling off") is a process of  programmed cell death that occurs in  multicellular organisms. [2] Biochemical events lead to characteristic cell changes ( morphology) and death. These changes include blebbing, cell shrinkage,  nuclear fragmentation,  chromatin condensation,  chromosomal DNA fragmentation, and global mRNA decay. Between 50 and 70  billion cells die each day due to apoptosis in the average human adult. [a] For an average child between the ages of 8 and 14, approximately 20 billion to 30 billion cells die a day." 5.

2. Altered inter-cellular communication

"Beyond cell-autonomous alterations, aging also involves changes at the level of intercellular communication, be it endocrine, neuroendocrine or neuronal ( Laplante and Sabatini, 2012;  Rando and Chang, 2012;  Russell and Kahn, 2007;  Zhang et al., 2013) (Figure 5C). Thus, neurohormonal signaling (eg, renin-angiotensin, adrenergic, insulin-IGF1 signaling) tends to be deregulated in aging as inflammatory reactions increase, immunosurveillance against pathogens and premalignant cells declines, and the composition of the peri- and extracellular environment changes, thereby affecting the mechanical and functional properties of all tissues." 6.

"Cells, as they age, show an increase in self-preserving signals that result in damage elsewhere. Altered intercellular communication with aging contributes to decline in tissue health.Like the decline in stem cell renewal, the age-dependent changes in intercellular communication are integrated effects of the other hallmarks of aging. In particular, senescent cells trigger chronic inflammation that can further damage aging tissues.The cdc42 GTPase pathway, in addition to the NF-κB pathway, has been shown to increase inflammation in senescent cells; in fact, knocking down cdc42 expression actually increases longevity in C. elegans. GTPases also integrate signals from cell-cell junctions, which may break down in aging tissues.At an organ system level, the aging hypothalamus drives changes in neurohormone signaling, which in turn affects food intake and metabolism. Since the hypothalamus also regulates sleep cycles, these changes can inhibit DNA repair, exacerbating the aging phenotype." 7.

3. Genomic Instability

Long Quote

"Genome instability (also "genetic instability" or "genomic instability") refers to a high frequency of  mutations within the genome of a cellular lineage. These mutations can include changes in nucleic acid sequences,  chromosomal rearrangements or  aneuploidy. Genome instability does occur in bacteria. [1] In multicellular organisms genome instability is central to carcinogenesis, [2] and in humans it is also a factor in some neurodegenerative diseases such as  amyotrophic lateral sclerosis or the neuromuscular disease  myotonic dystrophy.
The sources of genome instability have only recently begun to be elucidated. A high frequency of externally caused DNA damage [3] can be one source of genome instability since DNA damages can cause inaccurate translesion synthesis past the damages or errors in repair, leading to  mutation. Another source of genome instability may be  epigenetic or  mutational reductions in expression of DNA repair genes. Because  endogenous (metabolically-caused) DNA damage is very frequent, occurring on average more than 60,000 times a day in the genomes of human cells, any reduced DNA repair is likely an important source of genome instability.

The Usual Genome Situation

Usually, all cells in an individual in a given species (plant or animal) show a constant number of  chromosomes, which constitute what is known as the  karyotype defining this species (see also  List of number of chromosomes of various organisms), although some species present a very high karyotypic variability. In humans, mutations that would change an amino acid within the protein coding region of the genome occur at an average of only 0.35 per generation (less than one mutated protein per generation). [4]
Sometimes, in a species with a stable karyotype, random variations that modify the normal number of chromosomes may be observed. In other cases, there are structural alterations ( chromosomal translocations,  deletions ...) that modify the standard chromosomal complement. In these cases, it is indicated that the affected organism presents genome instability (also genetic instability, or even chromosomic instability). The process of genome instability often leads to a situation of  aneuploidy, in which the cells present a chromosomic number that is either higher or lower than the normal complement for the species."  8.

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Long Quote

Genomic Instability

"One common denominator of aging is the accumulation of genetic damage throughout life ( Moskalev et al., 2012) ( Figure 2A). Moreover, numerous premature aging diseases, such as Werner syndrome and Bloom syndrome, are the consequence of increased DNA damage accumulation ( Burtner and Kennedy, 2010), though the relevance of these and other progeroid syndromes to normal aging remains unresolved due, in part, to the fact that they recapitulate only some aspects of aging. The integrity and stability of DNA are continuously challenged by exogenous physical, chemical, and biological agents, as well as by endogenous threats, including DNA replication errors, spontaneous hydrolytic reactions, and reactive oxygen species (ROS) ( Hoeijmakers, 2009). The genetic lesions arising from extrinsic or intrinsic damages are highly diverse and include point mutations, translocations, chromosomal gains and losses, telomere shortening, and gene disruption caused by the integration of viruses or transposons. To minimize these lesions, organisms have evolved a complex network of DNA repair mechanisms that are collectively capable of dealing with most of the damages inflicted to nuclear DNA ( Lord and Ashworth, 2012). The genomic stability systems also include specific mechanisms for maintaining the appropriate length and functionality of telomeres (which are the topic of a separate hallmark; see below) and for ensuring the integrity of mitochondrial DNA (mtDNA) ( Blackburn et al., 2006;  Kazak et al., 2012). In addition to these direct lesions in the DNA, defects in the nuclear architecture, known as laminopathies, can cause genome instability and result in premature aging syndromes ( Worman, 2012)." 9.

4. Telomere Attrition

"As cells divide, the telomere ends of chromosomes get shorter. Eventually, the enzyme that adds telomeric repeat sequences, telomerase, gets silenced and the telomeres are too short for cells to divide. Shortened telomeres are associated with aging cells that are senescent.Telomeres at the ends of chromosomes, like all other sections of DNA, are prone to DNA damage, including double-strand breaks (DSBs). And unlike the rest of the chromosome, telomere DSBs aren’t fixed by the DNA repair pathway, as this would frequently lead to fused chromosomes and genomic instability. That’s why we have telomerase. However, telomerase expression is silenced in many adult cells, to curb rampant cell proliferation and tumorigenesis, and so telomeres get progressively shorter with age." 10.

Long Quote

"Accumulation of DNA damage with age appears to affect the genome near to randomly, but there are some chromosomal regions, such as telomeres, that are particularly susceptible to age-related deterioration ( Blackburn et al., 2006) ( Figure 2A). Replicative DNA polymerases lack the capacity to replicate completely the terminal ends of linear DNA molecules, a function that is proprietary of a specialized DNA polymerase known as telomerase. However, most mammalian somatic cells do not express telomerase, and this leads to the progressive and cumulative loss of telomere-protective sequences from chromosome ends. Telomere exhaustion explains the limited proliferative capacity of some types of in-vitro-cultured cells, the so-called replicative senescence, or Hayflick limit ( Hayflick and Moorhead, 1961;  Olovnikov, 1996). Indeed, ectopic expression of telomerase is sufficient to confer immortality to otherwise mortal cells without causing oncogenic transformation ( Bodnar et al., 1998). Importantly, telomere shortening is also observed during normal aging both in human and in mice ( Blasco, 2007a).
Telomeres are bound by a characteristic multiprotein complex known as shelterin ( Palm and de Lange, 2008). A main function of this complex is to prevent the access of DNA repair proteins to the telomeres. Otherwise, telomeres would be “repaired” as DNA breaks leading to chromosome fusions. Due to their restricted DNA repair, DNA damage at telomeres is notably persistent and highly efficient in inducing senescence and/or apoptosis ( Fumagalli et al., 2012;  Hewitt et al., 2012).  
Telomerase deficiency in humans is associated with premature development of diseases, such as pulmonary fibrosis, dyskeratosis congenita, and aplastic anemia, which involve the loss of the regenerative capacity of different tissues ( Armanios and Blackburn, 2012). Telomere uncapping and rampant chromosome fusions can also result from deficiencies in shelterin components ( Palm and de Lange, 2008). Shelterin mutations have been found in some cases of aplastic anemia and dyskeratosis congenita (Savage et al., 2008;  Walne et al., 2008;  Zhong et al., 2011). Various loss-of-function models for shelterin components are characterized by rapid decline of the regenerative capacity of tissues and accelerated aging, a phenomenon that occurs even in the presence of telomeres with a normal length ( Martínez and Blasco, 2010)." 11.

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Long Quote

"Telomeres shorten in part because of the end replication problem that is exhibited during DNA replication in eukaryotes only. Because DNA replication does not begin at either end of the DNA strand, but starts in the center, and considering that all known DNA polymerases move in the 5' to 3' direction, one finds a leading and a lagging strand on the DNA molecule being replicated.
On the leading strand, DNA polymerase can make a complementary DNA strand without any difficulty because it goes from 5' to 3'. However, there is a problem going in the other direction on the lagging strand. To counter this, short sequences of RNA acting as primers attach to the lagging strand a short distance ahead of where the initiation site was. The DNA polymerase can start replication at that point and go to the end of the initiation site. This causes the formation of Okazaki fragments. More RNA primers attach further on the DNA strand and DNA polymerase comes along and continues to make a new DNA strand.
 Eventually, the last RNA primer attaches, and DNA polymerase, RNA nuclease, and DNA ligase come along to convert the RNA (of the primers) to DNA and to seal the gaps in between the Okazaki fragments. But, in order to change RNA to DNA, there must be another DNA strand in front of the RNA primer. This happens at all the sites of the lagging strand, but it does not happen at the end where the last RNA primer is attached. Ultimately, that RNA is destroyed by enzymes that degrade any RNA left on the DNA. Thus, a section of the telomere is lost during each cycle of replication at the 5' end of the lagging strand's daughter.
However, test-tube studies have shown that telomeres are highly susceptible to oxidative stress. There is evidence that oxidative stress-mediated DNA damage is an important determinant of telomere shortening [25] Telomere shortening due to free radicals explains the difference between the estimated loss per division because of the end-replication problem (c. 20 bp) and actual telomere shortening rates (50–100 bp), and has a greater absolute impact on telomere length than shortening caused by the end-replication problem. Population-based studies have also indicated an interaction between anti-oxidant intake and telomere length. In the Long Island Breast Cancer Study Project (LIBCSP), authors found a moderate increase in breast cancer risk among women with the shortest telomeres and lower dietary intake of beta carotene, vitamin C or E [26] These results suggest that cancer risk due to telomere shortening may interact with other mechanisms of DNA damage, specifically oxidative stress.
Telomere shortening is associated with aging, mortality and aging-related diseases. In 2003, Richard Cawthon discovered that those with longer telomeres lead longer lives than those with short telomeres [27] However, it is not known whether short telomeres are just a sign of cellular age or actually contribute to the aging process." 12.

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5. Epigenetic alterations

Long Quote

"Epigenetics are stable heritable traits (or phenotypes") that cannot be explained by changes in DNA sequence. [1] The Greek prefix epi- (Greek: επί- over, outside of, around) in epigenetics implies features that are "on top of" or "in addition to" the traditional genetic basis for inheritance. [2] Epigenetics often refers to changes in a chromosome that affect gene activity and  expression, but can also be used to describe any heritable phenotypic change that doesn't derive from a modification of the genome, such as  prions. Such effects on  cellular and  physiological  phenotypic traits may result from external or  environmental factors, or be part of normal developmental program. The standard definition of epigenetic requires these alterations to be  heritable, [3] [4] either in the progeny of cells or of organisms.
The term also refers to the changes themselves: functionally relevant changes to the  genome that do not involve a change in the  nucleotide sequence. Examples of mechanisms that produce such changes are  DNA methylation and histone modification, each of which alters how genes are expressed without altering the underlying  DNA sequence. Gene expression can be controlled through the action of  repressor proteins that attach to  silencer regions of the DNA. These epigenetic changes may last through  cell divisions for the duration of the cell's life, and may also last for multiple generations even though they do not involve changes in the underlying  DNA sequence of the organism; [5] instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently. [6]
One example of an epigenetic change in  eukaryotic biology is the process of  cellular differentiation. During  morphogenesis,  totipotent  stem cells become the various  pluripotent  cell lines of the embryo, which in turn become fully differentiated cells. In other words, as a single fertilized egg cell – the  zygote – continues to  divide, the resulting daughter cells change into all the different cell types in an organism, including  neurons, muscle cells,  epithelium, endothelium of  blood vessels, etc., by activating some genes while inhibiting the expression of others. [7]
Historically, some phenomena not necessarily heritable have also been described as epigenetic. For example, epigenetic has been used to describe any modification of chromosomal regions, especially  histone modifications, whether or not these changes are heritable or associated with a phenotype. The consensus definition now requires a trait to be heritable for it to be considered epigenetic. [4]"  13.

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Long Quote

"As cells are exposed to environmental factors, they are subject to changes in their genome through epigenetic mechanisms. Such changes accumulate over time and have been correlated with the decline observed in aging cells. Epigenetic changes in aging include decreased methylation of H3K9 and H3K27, together with increased trimethylation of H4K20 and H3K4. In general, these changes are correlated with decreases in the amount of heterochromatin and consequent increases in chromosome fragility and transcriptional noise.Why do these molecular events occur? One theory is that age-associated DNA damage results in changes in the transcription of certain long noncoding RNAs (lncRNAs), such as KCNQ1OT1, PINT, and ANRIL, which then, in turn, regulate histone modifying enzymes. Whether this mechanism is directly relevant to aging remains to be proven." 14.

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6. Loss of Proteostasis

Long Quote

"Proteostasis, a portmanteau of the words  protein and  homeostasis, is the concept that there are competing and integrated  biological pathways within cells that control the  biogenesis, folding, trafficking and  degradation of proteins present within and outside the cell. [1] [2] The concept of proteostasis maintenance is central to understanding the cause of diseases associated with excessive  protein misfolding and degradation leading to loss-of-function  phenotypes, [3] as well as aggregation-associated degenerative disorders. [4] Therefore, adapting proteostasis should enable the restoration of proteostasis once its loss leads to pathology. Cellular proteostasis is key to ensuring successful development, healthy  aging, resistance to  environmental stresses, and to minimize homeostasis perturbations by pathogens such as  viruses. [2] Mechanisms by which proteostasis is ensured include regulated protein translation, chaperone assisted protein folding and protein degradation pathways. Adjusting each of these mechanisms to the demand for proteins is essential to maintain all cellular functions relying on a correctly folded proteome." 15.

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Long Quote

"As cells age, environmental stresses add up and mechanisms responsible for maintaining proper protein composition start to decline. Proteins lose their stability, autophagic processes start to fail, and misfolded proteins accumulate.Over the years, our bodies are subjected to many environmental inputs that put thermal stress, oxidative stress, and osmotic stress on our cells, causing misfolding of proteins. For example, free radicals present in polluted air have been identified as particularly noxious agents in this regard, contributing to multiple aging-related pathologies. In younger cells, micro- and macroautophagy pathways, together with the ubiquitin-proteasome system, take care of clearing these unfolded proteins. However, in aging cells, autophagy induction can be gradually compromised, and lysosomes become less efficient at eliminating the vesicles carrying this cellular waste.In a vicious cycle termed “inflammaging,” this decrease in efficient autophagy results in an increase in intracellular ROS, triggering the damage-sensing inflammasome to generate low levels of chronic inflammation, further accelerating aging." 16.

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7. Deregulated nutrient sensing


Long Quote

"Nutrient sensing is a cell's ability to recognize and respond to fuel substrates such as  glucose. Each type of fuel used by the cell requires an alternate pathway of utilization and accessory  molecules. In order to conserve resources a cell will only produce molecules that it needs at the time. The level and type of fuel that is available to a cell will determine the type of enzymes it needs to express from its  genome for utilization.  Receptors on the  cell membrane's surface designed to be activated in the presence of specific fuel molecules communicate to the cell  nucleus via a means of  cascading interactions. In this way the cell is aware of the available nutrients and is able to produce only the molecules specific to that nutrient type.

Nutrient Sensing in Mammalian Cells

A rapid and efficient response to disturbances in nutrient levels is crucial for the survival of organisms from bacteria to humans. Cells have therefore evolved a host of molecular pathways that can sense nutrient concentrations and quickly regulate gene expression and protein modification to respond to any changes. [1]

Cell growth is regulated by coordination of both extracellular nutrients and intracellular metabolite concentrations. AMP-activated kinase and mammalian target of rapamycin complex 1 serve as key molecules that sense cellular energy and nutrients levels, respectively.

The interplay among nutrients, metabolites, gene expression, and protein modification are involved in the coordination of cell growth with extracellular and intracellular conditions. [2]
Living cells use ATP as the most important direct energy source. Hydrolysis of ATP to ADP and phosphate (or AMP and pyrophosphate) provides energy for most biological processes. The ratio of ATP to ADP and AMP is a barometer of cellular energy status and is therefore tightly monitored by the cell. In eukaryotic cells, AMP-activated protein kinase (AMPK) serves as a key cellular energy sensor and a master regulator of metabolism to maintain energy homeostasis. [3]

Regulation of Tissue Growth Through Nutrient Sensing

Nutrient is a key regulator of tissue growth. The main mediator of cellular nutrient sensing is the protein kinase TOR (target of rapamycin). TOR receives information from levels of cellular amino acids and energy, and it regulates the activity of processes involved in cell growth, such as protein synthesis and autophagy. Insulin-like signaling is the main mechanism of systemic nutrient sensing and mediates its growth-regulatory functions largely through the protein kinase pathway. Other nutrition-regulated hormonal mechanisms contribute to growth control of modulating the activity of insulin-like signaling." 17.

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Long Quote

"Metabolic activities can put stress on our cells. Too much activity, and changes in nutrient availability and composition cause cells to age faster. Metabolism and its byproducts, over time, damage cells via oxidative stress, ER stress, calcium signaling, and mitochondrial dysfunction. Therefore, organisms depend on multiple nutrient sensing pathways to make sure that the body takes in just the right amount of nutrition – not too much, not too little. However, these damaging events also deregulate the nutrient-sensing molecules and downstream pathways. A misguided hypothalamus may signal for greater food intake, then, when the body doesn’t really require it. Age-related obesity, diabetes and other metabolic syndromes result. To make things even worse, obesity- and diabetes-related chronic inflammation, operating via JNK and IKK crosstalk, can deregulate nutrient sensing further.
Probably because so many interdependent pathways link metabolism to aging, these are the pathways that have received the most intense focus in the search for anti-aging therapeutics. There was much excitement in the last decade around resveratrol and caloric restriction, the effects of which have now been shown to be limited to mice and other model organisms. Today, intermittent caloric restriction (i.e., fasting) is the only intervention that has been shown to extend human lifespan." 18.

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8. Mitochondrial Dysfunction.

Long Quote

"The mitochondrion (plural mitochondria) is a double  membrane-bound  organelle found in all eukaryotic organisms. Some cells in some  multicellular organisms may however lack them (for example, mature mammalian  red blood cells). A number of unicellular organisms, such as  microsporidia,  parabasalids, and  diplomonads, have also reduced or transformed their mitochondria into other structures. [1] To date, only one eukaryote,  Monocercomonoides, is known to have completely lost its mitochondria. [2] The word mitochondrion comes from the  Greek μίτος, mitos, "thread", and χονδρίον, chondrion, "granule" [3] or "grain-like". Mitochondria generate most of the cell's supply of  adenosine triphosphate (ATP), used as a source of  chemical energy. [4]

Mitochondria are commonly between 0.75 and 3  μm in diameter[5] but vary considerably in size and structure. Unless specifically stained, they are not visible. In addition to supplying cellular energy, mitochondria are involved in other tasks, such as  signaling,  cellular differentiation, and  cell death, as well as maintaining control of the  cell cycle and  cell growth.[6]  Mitochondrial biogenesis is in turn temporally coordinated with these cellular processes. [7] [8] Mitochondria have been implicated in several human diseases, including  mitochondrial disorders, [9]  cardiac dysfunction, [10] heart failure [11] and autism. [12]

The number of mitochondria in a cell can vary widely by  organism,  tissue, and cell type. For instance,  red blood cells have no mitochondria, whereas  liver cells can have more than 2000; [13] [14] The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the  intermembrane space, the  inner membrane, and the  cristae and  matrix.

Although most of a cell's  DNA is contained in the  cell nucleus, the mitochondrion has its own independent  genome that shows substantial similarity to  bacterial  genomes.[15] Mitochondrial proteins (proteins transcribed from  mitochondrial DNA) vary depending on the tissue and the species. In humans, 615 distinct types of protein have been identified from  cardiac mitochondria, [16] whereas in  rats, 940 proteins have been reported. [17] The mitochondrial  proteome is thought to be dynamically regulated. [18]  19.

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Long Quote

"As cells age, their mitochondria start to lose their integrity due to the build-up of oxidative stress. Compromised mitochondrial function leads to a number of events, such as increased apoptosis induction, that correlate with aging.
Not only are mitochondria responsible for generating ATP, but they act as sensors of cellular distress, and are the first parts of the cell to send and respond to cell death signals. Since the 2013 publication of The Hallmarks of Aging, the role of mitochondria in regulating inflammation in response to metabolic change (via mitochondrial sirtuins) has received greater attention.
 Mitochondria send signals via calcium signaling and reactive oxygen species (ROS) to the NF-κB pathway, as well as via damage-associated molecular patterns (DAMPs) to the inflammasome, to activate “inflammaging.” In turn, proinflammatory molecules regulate mitochondria by causing decreases in mitochondrial membrane potential, a sign of poor cell health.
Melatonin signaling, on the other hand, affects the mitochondria positively, maintaining the integrity and function of these organelles. Melatonin decreases inflammation and maintains the efficiency of electron transport. Because of these powerful effects, maintaining melatonin levels via pharmaceuticals or by circadian regulation is an area of therapeutic interest. The future of mitochondrial dysfunction research will certainly include more genetic and epigenetic data around mtDNA, as well as their implications for innate immunity." 20.

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Long Quote

"Mitochondrial disease is a group of disorders caused by dysfunctional mitochondria, the organelles that generate energy for the cell. Mitochondria are found in every cell of the human body except  red blood cells, and convert the energy of food molecules into the  ATP that powers most cell functions.

Mitochondrial diseases are sometimes (about 15% of the time) [1] caused by mutations in the  mitochondrial DNA that affect mitochondrial function. Other causes of mitochondrial disease are mutations in genes of the  nuclear DNA, whose gene products are imported into the mitochondria ( mitochondrial proteins) as well as acquired mitochondrial conditions. Mitochondrial diseases take on unique characteristics both because of the way the diseases are often inherited and because mitochondria are so critical to cell function. The subclass of these diseases that have neuromuscular disease symptoms are often called a  mitochondrial myopathy." 21.

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9. Cellular Senescence

.Long Quote

"Cellular senescence is the point at which our cells stop dividing and growing due to damage or lack of necessary components. As cells age, they lose their ability to actively divide and start to undergo senescence.Senescence refers to a pause in the cell cycle, usually in response to damage. Young senescent cells are presumably cleared by the immune system, but in older tissues, they stick around, secreting harmful proinflammatory signals like IL-6 and IL-8 that damage our bodies further.As one would guess, the DNA damage and oxidative stress responses, via p53 and AMPK, induce senescence via the retinoblastoma pathway. And telomere shortening causes cells to stop replicating, reinforcing the senescent phenotype.The cell cycle regulator, p16, plays a still-mysterious role in aging-related senescence. Levels of p16 are more correlated to chronological age than are the levels of any other protein, but its precise regulation in the context of aging is not clear. Developmental signaling, such as by the Id proteins and microRNAs, is involved.The emerging theme is that it’s not enough to measure one or two biomarkers to unequivocally define the senescent state. To fully characterize aging cells, measure at least a few of the following phenotypic markers: secretory phenotype, beta galactosidase expression, proliferation, heterochromatic foci, flatness of morphology and chromatin alterations." 22.

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Long Quote

"Cellular senescence is the phenomenon by which normal  diploid  cells cease to  divide. In culture, fibroblasts can reach a maximum of 50 cell divisions before becoming senescent. This phenomenon is known as "replicative senescence", or the  Hayflick limit. [1] Replicative senescence is the result of  telomere shortening that ultimately triggers a  DNA damage response. Cells can also be induced to senesce via DNA damage in response to elevated  reactive oxygen species (ROS), activation of  oncogenes and cell- cell fusion, independent of telomere length. As such, cellular senescence represents a change in "cell state" rather than a cell becoming "aged" as the name confusingly suggests. Nonetheless, the number of senescent cells in tissues rises substantially during normal aging. [2]

Although senescent cells can no longer replicate, they remain metabolically active and commonly adopt an immunogenic phenotype consisting of a pro-inflammatory secretome, the up-regulation of immune  ligands, a pro-survival response, promiscuous gene expression (pGE) and stain positive for  senescence-associated β-galactosidase activity. [3] Senescence-associated beta-galactosidase, along with  p16Ink4A, is regarded to be a  biomarker of  cellular senescence. [4] Nonetheless, false positives exist for maturing tissue  macrophages and senescence-associated beta-galactosidase as well as for  T-cells p16Ink4A. [2] Senescent cells that grow attached to a solid substrate such as glass or plastic surface assume flattened appearance being thinner and having greater cellular and nuclear area. After staining nuclear DNA with a fluorescent dye the cellular imaging reveals a dramatic decrease in maximum pixel of the nuclear DNA-associated fluorescence accompanied by an increase in nuclear area; thus even a more remarkable decrease in maximal pixel to nuclear area ratio [5] A Senescence Associated Secretory Phenotype (SASP) consisting of inflammatory  cytokines,  growth factors, and  proteases is another highly characteristic feature of senescent cells. [6] SASP contributes to many age-related diseases, including type 2 diabetes and  atherosclerosis. [2] The damaging effects of SASP have motivated researchers to develop  senolytic chemicals that would kill and eliminate senescent cells to improve health in the elderly. [2] Healthy mice treated with senolytics have shown improved  cardiac and  vascular, function. [2] Removal of senescent cells in normal mice increased  health span as well as  life expectancy, [7]

The nucleus of senescent cells is characterized by senescence-associated  heterochromatin foci (SAHF) and  DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). [8] Senescent cells affect tumour suppression, wound healing and possibly embryonic/placental development and a pathological role in age-related diseases. [9]

The experimental elimination of senescent cells from transgenic  progeroid mice [10] and non-progeroid, naturally-aged mice [11] [12] [13] led to greater resistance against  aging-associated diseases.

Moreover, cellular senescence is not observed in several organisms, including  perennial plants,  sponges,  corals, and  lobsters. In those species where cellular senescence is observed, cells eventually become post- mitotic when they can no longer replicate themselves through the process of  cellular mitosis; i.e., cells experience replicative senescence. How and why some cells become post-mitotic in some species has been the subject of much research and speculation, but (as noted above) it is sometimes suggested that cellular senescence evolved as a way to prevent the onset and spread of  cancer. Somatic cells that have divided many times will have accumulated DNA  mutations and would therefore be in danger of becoming  cancerous if cell division continued. As such, it is becoming apparent that senescent cells undergo conversion to an immunogenic phenotype that enables them to be eliminated by the immune system. [14]"  23.

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Given the aforementioned hallmarks of aging at the molecular and cellular level and the definitions of said hallmarks, a general summation and definition of the aging process with regard to the same is appropriate.
The following long quote attempts to do the same.

Long Quote

"According to Harman [ 1], “Aging is the progressive accumulation of changes with time that are associated with or responsible for the ever-increasing susceptibility to disease and death which accompanies advancing age” and “the sum of the deleterious free radical reactions going on continuously throughout the cells and tissues constitutes the aging process or is a major contributor to it”. According to Hayflick [ 2], “The common denominator that underlies all modern theories of biological aging is change in molecular structure and, hence, function”.
While many authors believe that free radicals and oxidative stress play an insignificant role in aging (if any), it is hard to disagree with the clearly expressed view [ 2, 3,4, 5, 6, 7, 8,9,10] that aging is the generated by multiple causes damage to the structures and functions of the molecules, cells, organs, etc., of an organism. Such causes of aging include but are not limited to oxidative stress, glycation, telomere shortening, side reactions, mutations, aggregation of proteins, etc. In other words, it is the progressive damage to these structures and functions that we perceive and characterize as aging. This damage leads to development of pathological conditions and, as a consequence, to death. Hence, in agreement with the view expressed by Hayflick [ 3] and Holliday [ 4], we know the general cause of aging. This may be called the standard [ 10] or general (GTA) [11] theory of aging. Since it becomes more and more clear that aging is due to a significant number, or even a myriad, of causes [10, 11, 12, 13], the term unified theory of aging is applicable as well. Those who disagree with it have to clearly define what else aging could be from a mechanistic point of view, regardless of their more philosophical views about why we age and why and how life, aging, and death came into being.
Opinions concerning the possibility of counteracting aging range from very optimistic to very pessimistic views and are described in numerous papers and comments on such papers. Therefore, only few recent papers that discuss such matters are cited here [ 14, 15,16, 17].
There is a view that lifespan can be extended dramatically and some even suggest that practical immortality is possible and even achievable in the foreseeable future.
On the other hand, many are of the opinion that the human lifespan cannot be extended much beyond the current level, if at all, for the variety of reasons discussed in their papers. The view that there are myriad causes of aging, each one contributing insignificantly and thus too numerous to be protected against [ 12, 13, 18], seems especially pessimistic." 24.

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Thus, understanding the aforementioned, the following is a long quote which suggests means to address and prevent or ameliorate the aging process:

Long Quote

"The aging process is unquestionably complex. During an organism’s lifetime, aging cells undergo a plethora of changes and accumulate macromolecular damage. The aging phenotypes are manifested by the summation of alterations of many different signaling cascades. Here, we attempted to provide an overview of some of the major changes in aging cells, tissues, and organisms that have come to light in recent years through the lens of chromatin and epigenetic regulation. Genetic and environmental manipulations are unequivocally important to decipher the effect of any specific factor over the longevity process. It is becoming apparent mechanistically that many of those factors that do affect longevity act primarily through the modification of the epigenome. Undoubtedly, epigenetic influences over the aging process need to be incorporated into our current understanding of aging. Almost all classes of epigenetic alterations reported so far influence longevity pathways, adding further complexity to understanding the aging process. In summary, young healthy cells maintain an epigenetic state that promotes the formation of a compact chromatin structure and precise regulation of all the basic biological processes. However, aging cells experience alterations in all aspects of the chromatin landscape, DNA accessibility, and ncRNA production, until a threshold of altered gene expression and compromised genomic integrity is crossed, and the cells finally succumb to a permanent halt in progression through the cell cycle. The reversible nature of epigenetic mechanisms makes it possible to restore or reverse some of these phenotypes to attain more youthful cells.

Whereas some of the molecular changes during aging can be categorized as causal to aging, other changes simply accompany the aging process. However, when characterizing the causes or consequences of aging, one has to carefully dissect the experimental findings because most of the relevant pathways are interconnected, making it difficult to separate the effects of the pro-aging pathways from bystander pathways. A concerted effort should be made to establish the hierarchical relationship among the relevant pathways to understand the exact causal network of aging and to derive effective therapies to counteract the aging process and age-associated diseases. There is still a great deal to learn about this complex biological process. Current progress and extensive utilization of next-generation sequencing techniques are enabling us to analyze age-associated changes at ever-increasing resolution. However, simultaneous gain-of-function and loss-of-function studies in different organisms are also crucial to understanding and providing evidence of the causal effects of certain pathways, to move beyond correlation analyses. A tour-de-force analysis of the effect of individually deleting 4700 yeast genes on replicative life span was recently completed, finding 237 genes whose deletion extends life span  146). However, we clearly have a lot more to learn about the aging process because 30 of those genes are so poorly understood that they have yet to be given functional names.

The continued combination of functional studies and molecular analyses in different age groups, different organisms, and different tissue types will hopefully provide the details necessary to comprehend this evolutionarily conserved fundamental process and to facilitate the development of therapeutic interventions to counteract age-induced complications. A central concept that is emerging is to develop epigenetic drugs, or even an epigenetic diet, with the goal to improve not only a single disease state but also multiple disorders of aging. Although an intriguing idea, caution should be taken when determining the specificity of epigenetic therapies because of the interconnected nature of mechanisms that regulate epigenomic information. Thus, the major challenges that will dominate the field in the near future will be to achieve a hierarchical understanding of how epigenomics affects the aging process and to understand the long-term effects of therapeutic interventions on the epigenome in an aging individual, given the interconnectivity of the epigenetic mechanisms." 25.

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The End Replication Problem and Oxidative Stress

Long Quote

"Telomeres shorten in part because of the end replication problem that is exhibited during DNA replication in  eukaryotes only. Because DNA replication does not begin at either end of the DNA strand, but starts in the center, and considering that all known  DNA polymerases move in the 5' to 3' direction, one finds a leading and a lagging strand on the DNA molecule being replicated.
On the leading strand, DNA polymerase can make a complementary DNA strand without any difficulty because it goes from 5' to 3'. However, there is a problem going in the other direction on the lagging strand. To counter this, short sequences of  RNA acting as  primers attach to the lagging strand a short distance ahead of where the initiation site was. The DNA polymerase can start replication at that point and go to the end of the initiation site. This causes the formation of  Okazaki fragments. More RNA primers attach further on the DNA strand and DNA polymerase comes along and continues to make a new DNA strand.

Eventually, the last RNA primer attaches, and DNA polymerase, RNA nuclease, and  DNA ligase come along to convert the RNA (of the primers) to DNA and to seal the gaps in between the Okazaki fragments. But, in order to change RNA to DNA, there must be another DNA strand in front of the RNA primer. This happens at all the sites of the lagging strand, but it does not happen at the end where the last RNA primer is attached. Ultimately, that RNA is destroyed by enzymes that degrade any RNA left on the DNA. Thus, a section of the telomere is lost during each cycle of replication at the 5' end of the lagging strand's daughter.

However,  test-tube studies have shown that telomeres are highly susceptible to  oxidative stress. There is evidence that oxidative stress-mediated DNA damage is an important determinant of telomere shortening. [31] Telomere shortening due to free radicals explains the difference between the estimated loss per division because of the end-replication problem (c. 20 bp) and actual telomere shortening rates (50–100 bp), and has a greater absolute impact on telomere length than shortening caused by the end-replication problem. Population-based studies have also indicated an interaction between anti-oxidant intake and telomere length. In the Long Island Breast Cancer Study Project (LIBCSP), authors found a moderate increase in breast cancer risk among women with the shortest telomeres and lower dietary intake of beta carotene, vitamin C or E.[32] These results suggest that cancer risk due to telomere shortening may interact with other mechanisms of DNA damage, specifically oxidative stress.

Telomere shortening is associated with aging, mortality and aging-related diseases. In 2003, Richard Cawthon discovered that those with longer telomeres lead longer lives than those with short telomeres. [33] However, it is not known whether short telomeres are just a sign of cellular age or actually contribute to the aging process." 26.

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Long Quote

"During  chromosome replication, the  enzymes that duplicate DNA cannot continue their duplication all the way to the end of a chromosome, so in each duplication the end of the chromosome is shortened [6] (this is because the synthesis of  Okazaki fragments requires  RNA primers attaching ahead on the lagging strand). The telomeres are disposable buffers at the ends of chromosomes which are truncated during cell division; their presence protects the  genes before them on the chromosome from being truncated instead. The telomeres themselves are protected by a complex of  shelterin proteins, as well as by the RNA that telomeric DNA encodes ( TERRA).

Over time, due to each cell division, the telomere ends become shorter. [7] They are replenished by an enzyme,  telomerase reverse transcriptase." 27.

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Aging and Telomerase

Long Quote

"Telomerase replaces short bits of DNA known as  telomeres, which are otherwise shortened when a cell divides via  mitosis.

In normal circumstances, absent telomerase, if a cell divides recursively, at some point the progeny reach their  Hayflick limit. [19] which is believed to be between 50–70 cell divisions. At the limit the cells become senescent and  cell division stops. [20] Telomerase allows each offspring to replace the lost bit of DNA allowing the cell line to divide without ever reaching the limit. This same unbounded growth is a feature of  cancerous growth. [21]

 Embryonic stem cells express telomerase, which allows them to divide repeatedly and form the individual. In adults, telomerase is highly expressed only in cells that need to divide regularly especially in male sperm cells but also in epidermal cells, [22] in activated  T cell [23] and  B cell [24]  lymphocytes, as well as in certain  adult stem cells, but in the great majority of cases  somatic cells do not express telomerase." [25] 28.

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