The Hallmarks of Aging
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"The Hallmarks of Aging" is a peer-reviewed article published in the journal Cell by Carlos Lopez-Otın, Maria A. Blasco, Linda Partridge, Manuel Serrano, and Guido Kroemer in June 2013. Motivated by a review article entitled "The Hallmarks of Cancer" (published in the same journal in 2000  and updated in 2011), which had organized information from a vast body of research into the mechanisms underlying cancer, Lopez-Otin et al expressed the desire to do the same for the field of aging research, which had greatly accelerated in the three preceding decades. In their article the authors summarized the vast body of scientific literature relating to the aging process at the cellular level in a wide range of organisms, and identified 9 features of cellular physiology that they believed should be considered hallmarks of the aging process. To qualify as a hallmark, they proposed, a cellular property should be recognized to occur during normal organismal aging, there should be experimental evidence that increasing this property speeds up the aging process, and reducing this property should cause a recognizable improvement in health and lifespan.
As of May 2017, the article had been cited 1299 times in the scientific literature. 
The nine hallmarks that they proposed are:
Genomic instability As an organism ages, the likelihood of DNA damage accumulation increases. Additionally, the protective mechanisms that correct the damages become less efficient. The combined result of these two factors gives rise to a higher rate of DNA damage accumulation as time goes on, which then causes several of the phenotypes associated with aging. Futhermore, the nuclear lamina, a critical determinant of nuclear architecture and stability, also undergoes changes that result in genomic instability and have been observed to underly premature aging syndromes such as the Hutchinson-Gilford and the Néstor-Guillermo progerias.
Telomere Attrition At the end of replication, DNA polymerase is not able to replicate the ends of linear chromosomes, telomeres, where the RNA primer once was. This inability, leads to telomere shortening and the lose of genetic information.
Epigenetic Alterations Non-inheritable changes to the genome that do not change the sequence of the DNA itself are epigenetic alterations, which come in many forms. Epigenetic alteration has been shown to influence aging in humans and various model organisms.
Loss of Proteostasis The proteome, the entirety of proteins expressed in an organism, is significant to the function of the cell because proteins encode for essential structures and functional products. Proteostasis describes a state in which the expressed proteins in an organism are in homeostasis, marked by optimal regulation of protein expression, degradation, and their roles in signaling pathways.
Deregulated Nutrient Sensing Deregulated nutrient sensing is the loss of the cell's ability to detect and respond to nutrient levels, which affects metabolism by altering the balance between catabolism and anabolism leading to age-related obesity, diabetes, and other metabolic syndromes. Molecular pathways involved in nutrient sensing are conserved life span regulators and include caloric restriction, insulin and insulin-like growth factor signalling (IIS) and target of rapamycin (mTOR) signalling.
Mitochondrial Dysfunction Mitochondrial dysfunction is the creation of dysfunction through the creation of reactive oxygen species (ROS), the effect of hormesis, and the decrease in biogenesis in mitochondria. This often affects oxidative phosphorylation and the amount of energy in the body.
Cellular Senescence Cellular senescence is permanent growth arrest or the irreversible loss in the ability to divide in somatic cells. This serves a practical purpose as it prevents the division of damaged cells.
Stem Cell Exhaustion Stem Cells are responsible for the creation and maintenance of the differentiated tissues of an organism. Stem cell exhaustion occurs over time when the stem cells accumulate defects with reproductiona nd lose the ability to proliferate.
Altered Intercellular Communication Cells communicate with each other by three main mechanisms: neuronal, neuroendocrine, and endocrine. As aging progresses, changes or alterations in these communication mechanisms occur. 
- 1 Genomic instability: nuclear and mitochondrial DNA
- 2 Genomic instability: nuclear architecture
- 3 Telomere Attrition
- 4 Epigenetic Alterations
- 5 Loss of Proteostasis
- 6 Deregulated Nutrient Sensing
- 7 Mitochondrial Dysfunction
- 8 Cellular Senescence
- 9 Stem Cell Exhaustion
- 10 Altered Intercellular Communication
- 11 References
Genomic instability: nuclear and mitochondrial DNA
After Frederick Sanger developed techniques to rapidly sequence DNA in the 1970s, scientists were able to determine the causes of many health conditions and diseases that were previously unknown. Huntington's disease was one of the first genetic diseases to be mapped out and studied. The application of similar experimental techniques for other conditions/diseases revealed that the accumulation of DNA damage was common in biologically older organisms or organisms that demonstrated certain forms of accelerated aging. This damage can take many forms as a result of extrinsic or intrinsic factors. DNA damage that results in aging in multicellular organisms can be categorized into two groups based on the location of the DNA molecules- Nuclear DNA damage (located in the nucleus) and mitochondrial DNA (mtDNA) damage (located in the mitochondria). The Hallmark of Aging review paper identifies both forms of DNA damage as factors which accelerate aging. The paper groups these two forms of DNA damage under the sub-category of the "Genomic instability" hallmark. It is to be noted that, while both DNA and mtDNA are the same structurally, they have different repair mechanisms and are subjected to very different stressors. Nuclear DNA, in general, is considered better protected than its mitochondrial counterpart. The nucleus itself serves as a barrier between the stressors in the cellular environment and the DNA molecule. The nuclear DNA also has telomeres to protect its ends from degradation. Cells have evolved to have very efficient and damage specific DNA repair machineries, which are always employed in preserving the integrity of the DNA. Despite all these protective mechanisms, one of the greatest threats to nuclear DNA is aneuploides, (abnormal number of chromosomes) which is a product of miscommunication during the cell cycle. The presence of an irregular number of chromosomes can impact the competency of the Stem cells. Without functional stem cells, tissue renewal becomes hindered in the organism. Mitochondrial DNA lacks the different safety features which nuclear DNA has. It has not evolved efficient DNA repair mechanisms either. Additionally, the mtDNA molecule is constantly exposed to ROS, a byproduct of cellular respiration; this increases its likelihood of accumulating mutations. There is some debate over mtDNA mutations role in aging as mtDNA molecules demonstrate heteroplasmy. However, various studies have shown that individual aging cells have higher amounts of mtDNA mutation load and can even demonstrate heteroplasmy. At the time of publication of the review article, there was no research showing that a reduction in mtDNA mutation load can help extend lifespan.
Research done since the review publication
Since the publishing of “The Hallmark of Aging” review in 2013, many papers have been published on the subject, providing research which further elaborates on the role of the different hallmarks. With the recent advancement in bio-engineering tools and techniques, the field of genetics has especially progressed. This has provided us with a great deal of insightful and interesting information on the genomic instability hallmark.
Previously, there was no reliable simple invertebrate model organism that could be used to study the mtDNA mutation. However, in 2014, Leslie et al. published a paper showing that Drosophila melanogaster can serve as an effective model. The study also demonstrated that Reactive oxygen species are not responsible for the majority of the mtDNA mutations that translate into aging and aging related diseases.While their results are preliminary, and reproducibility of the data must be confirmed, it is definitely promising.
The recent technological progress has also provided scientists with the privilege of working with stem cells. This has furthered our understanding of inheritable diseases that are the result of germline mtDNA mutations. It was found that germline mtDNA mutations that lead to respiratory chain (RC) deficiencies are the products of downregulation in the transcription in mtRNAsas well as a reduction in the mtRNAs half life.
It was already known that mtDNA with extensive mutations will often undergo mitophagy; this is obstructed by proteins from the kinase family. Cells with inhibited rapamycin (mTOR) kinase activity supported this hypothesis by demonstrating a reduction in the number of mtDNA mutations, as well as production of more ATP.
Many neurodegenerative diseases are the result of mtDNA mutations. A great deal of research has been done to see how mtDNA mutations cause these conditions and what can we do reduce/diminish their effects. Some rare cases of Alzheimer's disease(AD) and Parkinson's disease (PD) are caused by mtDNA mutations. In general it was found that mtDNA mutation numbers increase,in the brain, in cases of both AD and PD( as well as Down Syndrome and Dementia). It was also found that patients with these conditions have reduced mtDNA mRNA levels, altered mtDNA copy number, and abnormal Aβ metabolism (aka Beta oxidation) in their brain tissues. Discovery of these common variables between the different diseases allows for “common genetic and pathophysiology explanation”, which can then be used to create for efficient treatments.
Dietary restriction is often used to reduce the effects of aging and related diseases. Research was done to examine the impact of dietary restriction on mice with Progeria (resulting in the accumulation of mtDNA mutations). For this, mutant mice were subjected to varying degrees of dietary restriction. It was seen that median and maximum lifespan increased by ~ 200%. Therefore, dietary restriction can aid in counteracting effects mtDNA damage related conditions.
The other theme that is constantly observed as our understanding of the different hallmarks increases, is that, these hall marks are often linked to one another and rarely exist as individuals. Telomere length and mtDNA instability are usually considered independent of one another. But recent research has demonstrated that telomerase, an enzyme that maintains of telomere length, is also responsible for responsible for responding to ROS related stress. In Tyrka et al. the scientists have demonstrated that mitochondrial proliferation and function declined in various tissues of mutant mice with severe telomere damage. The telomere dysfunction in some cases can turn on the p53 pathway.
Genomic instability: nuclear architecture
Before 2013 and based on the López-Otín et al. "Hallmarks of Aging" review, defects in nuclear architecture have been pointed to as a known cause of aging by various experts in the field. To understand the importance of nuclear lamins, it is key to also understand their role within the cellular nucleus. Nuclear lamins not only protect the DNA from outside factors, but it also has other important purposes, which include "providing a scaffold for tethering chromatin and protein complexes that regulate genomic stability."
Because nuclear lamins are such a major component of the nuclear envelope, their mutations can result major phenotypic effects on the organism. This is seen, for example, in accelerated-aging syndromes. The Hutchinson-Gilford progeria syndrome is caused by a mutation of the gene LMNA, which results in a buildup of the aberrant lamin A isoform named progerin. This mutated isoform results in misshapen nuclei.
Research done since the review publication
To understand the advances that have been made since the release of the López-Otín review, it is important to understand the role of the different types of lamins in the nuclear membrane, the results of protein malfunctions and mutations, the research that has been done in order to characterize the cause of the mutations and ultimately finding possible cures, and lastly the connection between the health and architecture of the nuclear lamina and other components that have been also known to also accelerate aging.
Lamins The nucleoplasm contains a complex, peripheral network known as the nuclear matrix. The peripheral region of the nuclear matrix is known as the nuclear lamina. This region is of high importance for the integrity of the cell because it interacts with inner nuclear membrane proteins, nuclear pore complexes, and peripheral chromatin. The nuclear membrane does not only contain lamins as its main or only component, it is also made up by other scaffolding proteins such as nuclear mitotic apparatus protein, known as NuMA, acting and several actin-binding proteins and less-known matrins or matrin-domain containing proteins.
A very important component of the nuclear envelope, in fact, are lamins; a type V intermediate filaments. In the human species, there are four types of lamins: A, B1, B2, and C. There are four types of lamin proteins, but only three genes in the human genome that encode for these four proteins. Through alternative splicing, the gene LMNA encodes for lamin A and lamin C; these two proteins are expressed in differentiated cells. The genes LMNB1 and LMNB2 encode for the B1 and B2 proteins, respectively, and at least one form of lamin B is expressed in every somatic cell.
Just like other proteins that perform specific roles in the cell, lamins undergo a series of post-translational modifications that allow them to be directed to the nuclear membrane including:
- Farnesylation: necessary for protein localization
- Advanced glycation end-products: seen in intima and media cells from large elastic arteries
- Phosphorylation of lamins
- Ubiquitylation and deubiquitylation: protein quality control in the endoplasmic reticulum
- Symoylation and desumoylation: a covalent and reversible lysine modification.
Even though lamins do have an important role in creating a stable nuclear envelope, it is not their only role within the cell. Besides proving a structural framework to the nucleoplasm, peripheral and nucleoplasmic lamins along with interacting protein partners and the many other nuclear membrane components play a number of important roles in the following:
- Chromatin organization
- DNA replication and repair
Hutchinson-Gilford Progeria Syndrome: A Disease Caused by Laminopathies Alterations to the LMNA gene through mutations have been known to cause cellular decline, which ultimately lead to severe phenotypic characteristics. Degenerative disorders caused by mutations of the lamin genes are known as laminopathies. Some of the phenotypes seen in patients diagnosed with nuclear morphological abnormalities include:
- Muscular dystrophies
- Premature aging diseases
Even with thorough understanding of the phenotypes seen in patients with defective lamin genes, the specific relationship between the gene mutation itself and different phenotypes is poorly understood. It has been difficult to determine this relationship because different mutations of the LMNA gene can lead to the same phenotype. Another factor that has intrigued research scientists is to try to understand why laminopathies have been seen to affect a single or few tissues, even when lamin A and lamin C are expressed throughout.
Back in 2003, two different laboratories published their findings relating the mutation of the LMNA gene to be the cause of the Hutchinson-Gilford Progeria Syndrome (HGPS). In this case, the research groups were able to determine the specific cause of this accelerated-aging syndrome. Both lamin A and lamin C are transcribed from the same gene through alternative splicing, which creates two very different lamins. Lamin C possesses five unique C-terminal residues, and lamin A is synthesized as a 664-residue prelamin A precursor that eventually becomes mature lamin A through a series of post-translational modifications.
Within these very important post-translational modification lies the mechanism that results in a faulty lamin A protein, known as progerin. It is not the mechanism itself that produces an erroneous lamin A protein, it is the mutation within the gene that does not allow the modifications to produce a properly mature lamin A protein.
A single base substitution within the LMNA exon 11 causes an activation of a cryptic splice site, which leads to an inframe deletion of 50 amino acids. Normally, the lamin A protein is farnesylated and carboxymethylated. In case of the abnormal lamin A, within the 50 amino acids that were deleted was a site for endoproteolytic cleavage, which renders the lamin permanently farnesylated and carboxymethylated. This single-base mutation has been seen to cause cellular decline through a series of mechanisms including epigenetic changes, telomere shortening, DNA repair defects, misregulated gene expression, and the characteristic phenotype of abnormal nuclear morphology.
This abnormal lamin A is known as progerin which is not only seen in accelerated-aging syndrome, but also in senescent cells and cells from old individuals. This suggests that the production of progerin could a factor in aging.
Technology Advancements for the Study of Lamins To be able to study lamins, these structures have to be purified from the rest of the other components that share the same nuclear environment without causing artifacts. Even though lamins are part of such complexes structures, new methods have been introduced since 2013 that facilitate laminal purification in order to understand laminal cellular organization.
For example, Ce-lamin was ectopically expressed ex vivo in Xenopus laevis oocytes and then purified using minimal disruptive methods. The samples were then visualized using cryo-electron tomography. Analysis showed that Ce-lamin "assembles into flexible protofilaments that interacts with each other and exhibit a diameter of 5–6 nm. These data show that protofilaments are the basic assembly units in vivo and that they can assemble into thicker, higher order, filaments. Therefore, the 10 nm intermediate filament-like lamin filament structure represents only one form of assembly out of several assembly possibilities."
Even though significant movement toward a future of full understanding of how lamins interact with each other and the other components of the nuclear lamina, there is still ample room for improvement. Gruenbaum et al. argue that new tools in "biochemistry, imaging and structural techniques, such as high-resolution detectors for cryo-ET and super-resolution fluorescence microscopy, in combination with established methods, will likely provide unprecedented view of lamins in the near future."
Research Articles Published in Relation to Laminopathies: 2013-2017 Since 2013, there has been an abundant number of research laboratories trying to further characterize the relationship between disruptions of the nuclear lamina and its role in physiological aging in order to develop treatments for various, known diseases.
Chemical inhibition of NAT10 corrects defects of laminopathic cells This article was published on May 2, 2014 in the Science Magazine journal which is under the American Association for the Advancement of Science (AAAS) organization.
In this article, the researchers aimed the study at finding a way to fix the misshapen nuclei and altered chromatin organization characteristic of diseases associated with cancer and laminopathies, especially the Hutchinson-Gilford Progeria Syndrome (HGPS).
The authors understood that a misshapen nucleus can be detrimental to the cell because it renders the cell fragile and also because such altered shape leads to a downstream effects on chromatin structure, gene expression, DNA replication, or DNA repair. Therefore, their hypothesis was that by restoring nuclear shape, this would improve chromatin structure and ameliorate all of the other side effects, and ultimately see an improved organismal phenotype.
To start the experiment, Homo sapiens bone osteosarcoma (U2OS) cells were transfected by small interfering RNA (siRNA) that silenced the LMNA gene (siLMNA). This change in the cell's genome was seen to cause nuclear shape defects, global chromatin relaxation, and increased nuclear area when compared to a healthy nucleus.
The authors turned their attention into finding out the specific acetyltransferase responsible for the translation of the erroneous copy of the LMNA gene. Based on compound screening, they found that 4-(4- chlorophenyl)-2-(2-cyclopentylidenehydrazinyl) thiazole, named compound (1), was able to restore nuclear circularity and global chromatin compaction through its inhibition of a lysine acetyltransferase (KAT).
A couple of factors of compound (1) made it a particularly important molecule to study including:
- Molecule (1) was identified in Saccharomyces cerevisiae as a GCN5 network inhibitor, but its effects were independent of this network.
- Molecule (1) was seen to improve the nuclear morphology of several cancer cell lines: this indicated that the effects of the compound were not specific to siLMNA cells.
From compound (1), the researchers used "click-chemistry," a method that taggs a small molecule to a specific drug-associated protein, in order to make it easier to find out the acetyltransferase targeted by molecule (1). From this experiment, the scientists found that N-acetyltransferase 10 (NAT10) was the target of compound (1).
Because of its instability, compound (1) was used to create a homolog named "Remodelin." Remodelin was then tested on siLMNA and HGPS-derived patient cells, and it was found to produce the same results as compound (1) regarding its ability to remodel nuclear shape.
This experiment used chemical, cellular, and genetic approaches to try to find a possible cure to laminopathies, using HGPS-cells as a real-world example for future applications.
Disruption of PCNA-lamins A/C interactions by prelamin A induces DNA replication fork stalling This article was published online on September 27, 2016, in the journal Nucleus.
The article did not look into lamin gene mutation; its focus was the accumulation of prelamin A, the precursor to mature lamin A before it has undergone important post-translational modifications, and its effects in cellular health.
According to the authors, evidence suggests that prelamin A accumulates in cells as an organism ages, specifically in vascular smooth muscle cells. The accumulation of prelamin A has cytotoxic effects, including DNA damage. The relationship between the accumulation of the protein with DNA damage is poorly understood, however.
It is known that lamins interact with components of the DNA replication machinery, including the sliding-clamp proliferating cell nuclear antigen (PCNA). PCNA provides a sliding clamp for DNA polymerase δ. Its presence is also important in maintaining the connection between the polymerase and the DNA. The interactions between lamins and PCNA actually serve the purpose of positioning PCNA on nuclear chromatin. If the LMNA gene is mutated, then this interaction between PCNA and lamins is compromised which results in reduced binding affinity.
The authors used U2OS cells to test how expression of prelamin A affected the PCNA.
It was found that the expression of prelamin A caused mono-ubiquitination of PCNA and increased formation of Pol η foci. These two results are indicative of DNA replication fork stalling. The authors concluded that prelamin A was involved in the stalling of the DNA replication fork, and thus of the whole process of DNA replication. The study finds that for the first time prelamin A was shown to directly induce DNA damage via DNA replication.
The overall conclusion of the researchers stated that the DNA replication fork stalling by prelamin A eventually induced double strand breaks of the DNA which hinders the ability of the cell to maintain homeostasis. When focused on cardiovascular tissue, the authors presented a possible relation between prelamin A accumulation and natural aging.
Cofilin Regulates Nuclear Architecture through a Myosin-II Depeasdfsdfssddhanotransduction Module This research article was published on January 19, 2017, on the journal Scientific Reports.
The authors wanted to explore the mechanism that regulates nuclear shape, and they found a relationship between F-acting cytoskeletal organization and nuclear morphology.
The study finds that cofilin/ADF-family-F-acting remodeling proteins are essential for normal nuclear architecture in different cell types.
By using siRNA to silence the gene encoding Cofilin/ADF, the researchers saw a series of striking nuclear defects including shapes, nuclear lamina disruption, and reductions to peripheral heterochromatin.
From these observations, the experiments focused on how the Cofilin/ADF modulates other components in the cellular nucleus. It was found that Cof/ADF modulates myosin-II activity through competitive inhibition for binding to F-actin.
The researches concluded that nuclear shape was not completely controlled by general cytoplasmic F-actin organization, but through intracellular actomyosin forces.
This study thoroughly concludes by stating that a faulty cofilin and nucleo-cytoskeletal mechanocoupling can result in abnormal nuclear shape, and the importance of this finding for the field of laminopathies research.
Studies of the Correlations between the Nuclear Architecture Hallmark of Aging to Other Hallmarks From the Same Review The task of understanding the mechanisms underlying physiological aging is no simple one. This is because biological systems are part of complex networks that influence each other in direct and indirect ways. Therefore, to understand how the many mechanisms are connected is a large portion of current studies trying to determine the factors that underlie physiological aging.
A review article published on April 11, 2016, in the journal Nucleus focused on the relationship between mammalian telomeres and lamins.
According to the review, numerous aspects at the biochemical, cellular and organismal level suggest that telomeres and lamins are more related than previously thought.
Telomeres interact with lamins through telomeric proteins and telomere-associated factors. If an alteration happens to either or both of the structure, cellular decline is bound to be observed.
Through this interaction, the review determines that the study of cellular senescence, a hallmark of aging, can also be brought into the complex mix of interacting factors that cause aging.
The authors state that the most striking link between lamins, telomeres, and senescence is the ability of telomerase reverse transcriptase (TERT) to rescue senescence induced by both lamins and telomere alterations.
Upon damage to the laminal and telomere structure of the cell, it also been seen that stem cell exhaustion is induced.
This review is an example of how the scientific community is starting to develop more interdisciplinary approaches to understanding the mechanisms of aging.
At the end of replication, DNA polymerase is not able to replicate the ends of linear chromosomes, telomeres, where the RNA primer once was. This inability, leads to telomere shortening and the lose of genetic information, unless the cell is one of few which has the enzyme telomerase, which accounts for the loss from DNA polymerase. For cells without telomerase, DNA polymerase’s incapability to replicate the telomeres leads to a shortening of the chromosome over time. A lack of telomerase can also lead to many diseases, all having to do with a loss of regenerative capabilities. Due to this observation, telomere attrition has been strongly linked to aging, as described in “The Hallmarks of Aging” review article. It has also been seen that telomeres endure much more age-related DNA damage than other parts of the chromosome, which further contributes to telomere attrition, strengthening its role in aging.
Besides telomerase, another protective measure that telomeres possess is the shelterin complex, which binds to the telomeres, preventing neighboring chromosomes from being joined due to DNA damage repair mechanisms. This complex is of importance because a malfunction of this complex can lead to accelerated aging.
Studies have shown that the length of telomeres correlates with the lifespan of an organism. Mouse models with shortened telomeres had a shorter lifespan and mice with longer telomeres had a longer lifespan. Also, using mouse models, it was seen that if telomerase was active, this would lengthen the life of the mice for a certain period, therefore making a positive connection between telomerase levels and overall lifespan. With this experimental knowledge, telomere attrition is considered a hallmark of aging.
Research done since the review publication
There has been a significant amount of research regarding telomeres and aging since the "Hallmarks of Aging" review paper. Many researchers have focused on determining whether other organisms, such as birds, rodents and fish have shortened telomeres as they age. One example is the zebrafish, which was found to have shorter telomeres in key tissues which lead to aging, both within the tissue but also the organism as a whole. Another example was performed in plants, Arabidopsis, and the authors found that telomere dynamics were connected to meristem activity and continuous growth of the plants. Contrasting from humans, plants and zebrafish, the edible dormouse had elongated telomeres as they aged, which was accredited to a reproduction strategy which makes them not reproduce during times of low food. The mechanism by which the edible dormouse elongates its telomeres has not been excessively looked at since the present paper is recent.
It was also found that environmental factors can play a role in the shortening of telomeres. A study was done with arctic breeding kittiwakes studying the effect of oxychlordane on telomere length. Oxychlordane belongs to a type of chemicals which are persistent organic pollutants (POPs). Arctic breeding kittiwakes were used because the arctic is where POPs get absorbed into the marine life and get eaten by the seabirds.The authors discovered that oxylchlordane was strongly associated with shorter telomeres in this particular bird, but only in females. This sex-selectivity could possibly be due to the increase in susceptibility to oxychlordance by egg production.
Telomere dysfunction/deficiency has been linked to a number of diseases including pulmonary fibrosis. A recent article showed the mechanism by which telomeres affect and lead to the onset of pulmonary fibrosis via the Trf1 gene in mice.
Epigenetic alterations consists of a variety of changes that can occur in the genome. Unlike DNA mutations, these epigenetic alterations are reversible. Examples of epigenetic alterations include DNA methylation, histone modification, and chromatin remodeling. Many enzymes like methyltransferases, (de)methylases, (de)acetylases, and heterochromatin remodeling proteins are under investigation for their ability to alter DNA and their potential to influence lifespan.
Investigation into methylation patterns of the genome during aging suggest that there are various hypermethylated regions that show up through the aging process, which refutes the previous belief that aging was due to a general demethylation of the genome. Based on the 2013 review The Hallmarks of Aging, there has been no definitive experimentation done correlating increased lifespan to methylation patterns of the genome.
Manipulation of histones by underexpression of the modifying enzymes H3K4 and H3k27 in nematodes and flies, respectively, results in a decrease in longevity, suggesting that histone modification may have direct correlation to lifespan. In vertebrates, a class of proteins called sirtuins, also found in red wine, improve health during aging, rather than increasing longevity, by stabilizing the genome, aiding glucose homeostasis, and deacetylation of mitochondrial proteins.
Through chromatin remodeling, different parts of the genome are exposed and available for transcription, or for methylation. In a study done with flies underexpressing a heterochromatin remodeling protein 1a, lifespan and health was greatly reduced. Chromatin remodeling may also have a direct effect on telomere building and length, which is another hallmark of aging.
Research done since the review publication
DNA Methylation As the genome becomes methylated or demethylated, the organization of chromatin can be effected and/or genes can be repressed or activated which may change the proteome of the cell which may result in physiological aging. Current novel research into the effects of DNA methylation by Horvath, et al. done in a 2014 published journal article predicts age based on the level of DNA methylation as a function of BMI units, suggesting that obesity causes epigenetic alterations of the DNA that accelerate physiological aging in liver tissue. In addition to this, it has been shown that other environmental factors other than diet, like smoking habits and exercise habits, can affect epigenetics in a way that results in accelerated physiological aging. Furthermore, decreased epigenetic regulation by methylomes has been determined to be reduced with age in human epidermal tissue samples by statistical analyses. Research done by Borman, et al. in 2016 also suggests, in accordance with Horvath, that DNA methylation can be a predictor of age, but in skin tissue. Both sets of research make use of microarrays and public datasets, current technology that allows for the analysis of thousands of methylation sites among thousands of individuals, genes, and tissues. Histone Modification In the sub-category, histone modification, of epigenetic alterations, more current research by Zhang, et al. in a 2015 published article shows that nuclear structure and epigenomic organization can be altered by a lack of functional WRN gene, associated with the premature aging disorder Werner syndrom, shown by a down-regulation of the H3K9me3 heterochromatin mark. The WRN protein associates with heterochromatin proteins and nuclear lamina proteins that control the structure of the genome. It is well known and characterized that disorder of nuclear lamina proteins is also a hallmark of aging. In the disease Hutchinson-Gilford Progeria Syndrome (HGPS) characterized by severe premature ageing phenotype, the H3K9me3 and H3K27 are, again, shown to be downregulated. However, Arancio, et al. also notes that the heterochromatic marker H4K20me3 is upregulated in HGPS cells, which marks telomeric heterochromatin, and that "histone H4K16 hypoacetylation is associated with premature senescence." These research highlight the interconnection between different types of epigenetic alterations and between other hallmarks of aging, like telomere shortening, which remind us that aging is a complex process that most likely is not caused by any single phenomenon.
Chromatin Remodeling As exemplified above, histone modification is intimately associated with chromatin remodeling, since the main units of chromatin are histone proteins on which the DNA is wrapped around to create dense compact chromatin, eventually leading to the most condensed and readily divisible form: chromosomes. Recent study of the effects of chromatin remodeling shows that loss of local interactions between chromatin and TADs (topologically associating domains) or LMNB1 leads to a change in the physical compactness of the DNA unique in senescent cells. LMNB1 is one of four lamin proteins found in the nuclear architecture, and alternative splicing of the mRNA of these genes can lead to defective lamins, causing laminopathies resulting in premature physiological aging.
Loss of Proteostasis
Loss of proteostasis, as a result of aging, causes defects in the machinery that regulate the proteome of an organism. This can result in a variety of harmful effects on the cell, such as the accumulation of defective proteins, proteins that are not effectively expressed, and defective chaperone proteins. Typical protein unfolding is caused by heat shock, ER stress and oxidative stress. However, the cell has specific mechanisms by which it addresses the natural aggregation of mis-folded or non-functional proteins. These mechanisms become less regulated over time, and causes the aggregation of non-functional proteins that contribute to aging in the model organism. The three main mechanisms that prevent the loss of proteostasis are the ubiquitin-proteasome system, the autophagy-lysosomal system, and chaperone mediated protein folding. The autophagy-lysosomal system, and ubiquitin-proteasome system support proteostasis by facilitating the degradation of non-functional proteins. Autophagy is a process by which an organism not only carries out the degradation of faulty proteins, but also accelerates protein turnover. Chaperone proteins facilitate protein refolding, in order to ensure protein stability and quality.
Ubiquitin-Proteasome System Essentially, the ubiquitin proteasome system utilizes a variety of enzymes, and ubiquitin tagging in order to direct non-functional proteins to be degraded in the proteasome complex. The main enzymes present in this system are known as E1, E2, and E3. E2 is the most central enzyme in this system because it carries out ubiquitin tagging, on proteins that are non-functional. Proteins tagged with ubiquitin are guided into the proteasome complex to be degraded. This process process prevents the aggregation of non-functional proteins, which contributes to aging.
Chaperone Mediated Protein Folding Chaperones are a group of proteins that influence covalent protein folding, and protein unfolding in order to ensure the stability of the proteome. Chaperone proteins have a variety of roles in the cell which include, preventing aggregation, ensuring that proteins follow a protein folding pathway, and facilitating the process of folding newly synthesized proteins.
Autophagy-Lysosomal System Authophagy is the degradation of damaged or non-functional material in the cell, such as organelles or proteins. The process of autophagy is carried out by autophagosomes, and is typically triggered when the cell experiences stress. Autophagy can be carried out in three distinct methods known as chaperone mediated autophagy, macroautophagy, and microautophagy. All three of these methods result in the damaged cell material from the cytoplasm being lead to the lysosome lumen for degradation.
Research done since the review publication
The hallmarks of aging outlines the mechanisms by which the cell maintains optimal protein regulation, which include chaperone-mediated protein folding, the autophagy-lysosomal system, and the ubiquitin-proteasome system. In the hallmarks of aging there is a figure that outlines three endogenous and exogenous forms of stress that initiate protein unfolding, and how subsequent pathways involving the previously stated mechanisms can be affected by the aggregation of mis-folded and non-functional proteins.
In one study performed after 2013, a transgenic mouse model was constructed to have a defects in chaperone mediated autophagy, specifically in liver tissues. With this induced defect, caused by the deletion of L2A in the liver, it was clear that there was a decline in proteostasis in the model organism, due to vastly reduced levels of chaperon mediated autophagy. Inducing the decline of chaperone-mediated autophagy in the hepatic system of mice, caused greater vulnerability to oxidative stress, the inability to metabolize drugs, as well as reduced hepatic function overall. Thus, compromising the chaperone medediated protein folding in this tissue has greater effects on the overall function of this tissue, due to non-functional proteins, that code for essential functional products in the cell.
Other studies in aging and longevity have taken a different approach, and seek to preserve or maintain proteostasis in a model organism to demonstrate how proteostasis influences longevity. Experimental methods can be used to induce moderate heat stress, a natural exogenous occurrence in the cell, in order to trigger autophagy in a model organism that normally experiences a decline in the autophagy-lysosomal system with age. Many studies talk about hormesis, a method which induces moderate quantities of stress to the organism, which can have beneficial effects, such as increased longevity. In this study exposing the model organism, C. elegans, to hormetic heat stress induces autophagy, which becomes less regulated with age. Inducing autophagy resulted in greater stress resistance and longevity in the model organism, as a result of maintaining proteostasis via the autophagy-lysosomal system.
In another study a transgenic fruit fly model was developed to over express FOXO, in order to improve the functionality of the ubiquitin-proteasome system with age. Moderate over-expression of dFOXO in heart tissue of D. melanogaster, resulted in improved protein maintenance mainly in the ubiquitin-proteasome system, but also in the autophagy-lysosomal system. Over-expressing this gene had downstream effects on the ubiquitin-proteasome system, which allowed it to work at an optimal level, even in aged D. melanogaster. Greater protein regulation, caused by moderate over-expression of dFOXO in the heart tissue, significantly improved overall function of this tissue in aged fruit flies.
An abundance of aging research focuses on the investigation of protein aggregates, in order to quantify aggregation in specific tissues, and how it affects tissue function. A study conducted on the skeletal muscle in humans focuses on quantifying protein aggregates in tissue samples obtained from a biopsy in order both old and young subjects. The skeletal muscle samples in older subjects contained 2 fold more protein than the younger subjects. Given these quantifications, this study was applied to skeltal muscle in C. elegans to see the true effects of aggregation on skeletal muscle. It was clear that protein aggregation, a result of loss of proteostasis, caused decreased muslce mass, and decreased mobility.
Overall, these recent studies seek to understand the greater implications of dysfunction in the proteome, by examining the machinery that maintains optimal protein regulation in the cell. These mechanisms are meant to promote protein turnover, and eliminate the buildup of non-functional proteins, because optimal protein function is essential to the overall health of the organism, given that proteins have diverse roles in the cell.
Deregulated Nutrient Sensing
Deregulated nutrient sensing is when the body’s detection and response to fuel, such as glucose, becomes unregulated. The review focuses on the insulin and insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway, mTOR, AMPK (AMP-activated protein kinase), and sirtuins. Dietary restriction (DR) was also shown to extend longevity.
The IIS pathway and its FOXO and mTOR targets have been well-maintained in evolution. Reduced signaling strength along this pathway has been shown to increase longevity in worms, flies, and mice, as well as mimic some of the effects of DR. However, there seemed to be a required balance where downregulation of the IIS pathway led to longevity, but extremely low levels of its components led to increased aging or even death. In worms and flies, transcription factor FOXO has shown to be relevant to aging, but little was known about the four FOXO members in mice. However, mice with increased levels of tumor suppressor PTEN have displayed downregulation of the IIS pathway, increased energy spending, and increased longevity.
The mTOR kinase senses high nutrient levels and anabolism through the presence of amino acids. Downregulation of mTORC1, one of the two kinases of mTOR, mimics the effects of DR and increases longevity in yeast, worms, and flies, even with normal regulation of mTORC2. Knocking down a component of mTORC1, such as S6K1 was also sufficient to extend lifespan. The addition of rapamycin also increased longevity in mice. Despite the beneficial effects of mTOR inhibition, side effects exist, such as cataracts, insulin resistance, and deficiencies in wound repair. AMPK senses low nutrient levels and catabolism through the presence of AMP. Increased activity of this kinase increases lifespan and downregulates mTORC1, extending these benefits. Sirtuins also sense low energy levels through NAD+ levels. Likewise, increased sirtuin levels activate metabolic responses and are part of a positive feedback loop with AMPK.
Research done since the review publication
Since the publication of the “Hallmarks of Aging” review, the components of deregulated nutrient sensing have been investigated further. More information in regard to pathway components are currently being researched with the hope of better understanding the causes of deregulated nutrient sensing and its contribution to the aging phenotype. More treatments can be developed to protect against age-related disease when new information is discovered. A common trend is the interconnection of the hallmarks. The more research that is done, the more connections seem to be made. Below are some advances made since the "Hallmarks of Aging" review. Specifically, the mechanisms underlying caloric restriction and the AMPK pathway were further investigated. Additionally, sirtuins, briefly mentioned in the review, and sestrins, not mentioned at all, were found to play a large role in deregulated nutrient sensing.
Caloric Restriction The processes behind the effects of caloric restriction on longevity have been further investigated. Caloric restriction is defined as consuming fewer calories, but not to the extent of malnutrition. The lifespan benefits are conserved across species from yeast to primates. It has been shown to reduce visceral fat, which produces increased metabolic flexibility. Visceral fat promotes inflammation and dibetes, which explains why its reduction is beneficial.
Caloric restriction also triggers autophagy, which supports healthy organelles, stem cell function, and immunology. It does so by utilizing nutrient sensors, such as SIRT1, AMPK, and MTORC1 to activate autophagic responses, FOXO1, and preserve telomerase. In particular, activating FOXO has been shown to play a role in the telomere attrition hallmark through improving telomerase activity, showing again how the hallmarks are connected.
Calorie restriction mimetics are a new concept to alleviate the side effects of caloric restriction, such as anemia and loss of muscle mass. For example, macronutrient intake has been shown mimic the effects of caloric restriction in mice fed ad libitum. Specifically, carbohydrates and protein levels dictate dietary intake. The longest lifespans were observed in mice with low-protein and high carbohydrate diets. Even though these mice were fed ad libitum, they showed increased lifespan due to changes in diet. Specifically, the low protein and high carbohydrate diet inhibits mTOR, which extends lifespan. This highlights the importance of macronutrient intake on lifespan and healthspan.
AMPK and Adenosine Derivatives Maintenance of adenosine derivatives (AMP, ADP, and ATP) at constant levels is central to cell metabolism and energy homeostasis. AMP and ADP are precursors to ATP, which tranports energy throughout the cell for metabolism. AMP biosynthesis was previously shown to be involved with increasing lifespan in yeast, but no work had been done with multi-cellular organisms. Likewise, AMP:ATP ratios and increased AMPK activity were shown to be predictive of lifespan in C. elegans, but had yet to be researched in more complex organisms. AMP biosynthesis, adenosine nucleotide ratios, and AMPK were more recently shown to be determinants of lifespan in Drosophila melanogaster. The addition of adenine to the diet of flies reduced lifespan by interfering with the positive effects of dietary restriction. Manipulating dietary adenine could alter metabolism to influence lifespan. Sirtuins Sirtuins are a family of proteins that are involved in a variety of signalling pathways. In particular, one member of this family, sirtuin 1 (SIRT1), is an NAD+ deacetylase. Activation of SIRT1 has been shown to improve metabolism and alleviate some age-associated phenotypes. SRT1720 activates SIRT1. Dietary supplementation of SRT1720 in mice fed on a standard diet improved both lifespan and healthspan.
SIRT1 was also activated by temperature reduction in fish. In some organisms, temperature reduction was shown to increase lifespan. However, little was known about the mechanism. Researchers found that temperature reduction stimulates the synthesis of SIRT1 and FOXO3A/FOXO1A, which are both downstream regulators of the IIS pathway. Additionally, they found that reactive oxygen species (ROS), which causes damage that leads to aging, was reduced with temperature reduction, furthering the connections between causes of aging.
Sestrins Sestrins are a family of conserved proteins activated by stress. They are important in maintaining metabolic homeostasis by reducing ROS and regulating the AMPK and mTOR pathways. Obese mice deficient in sestrins were shown to get diabetes sooner than mice without the deficiency. Mice lacking sestrin 3 (Sesn3) in the liver displayed metabolic disorders, such as insulin resistance and glucose intolerance, typically seen with accelerated aging. Conversely, Sensn3 transgenic mice were protected against these disorders, even with a high-fat diet. These results were the effect of sestrin 3 activating mTORC2.
The mitochondrion is an organelle inside of a eukaryotic cell. The mitochondrion has two ogranelle membranes. In these membranes the electron transport chain (ETC) is contained. This electron transport chain connects the shuttling of electrons across the inner membrane with the transfer of protons to create a proton gradient. This gradient allows for the synthesis of adenosine triphosphate (ATP) which is crucial for the cell to survive and is a source of energy.
Miochondria have their own set of DNA called mtDNA. This form of DNA is different then the DNA in the cell's nucleus because it is only inherited from the mother. Mutations in the DNA can cause dysfunction in proteins that help in the electron transport chain. ROS can be another source of dysfunction when they are at high levels. However, newer research has seen a correlation between more ROS and less aging and dysfunction which might be beneficial to the organism.
Although it was previously thought that outside treatments that were mildy toxic had a harmful effect on organelles, it might actually be beneficial to them. This is called mitohormesis and may actually cause an increase in cellular functioning. Therefore, treatments that would originally damage the cell and the mitochondria and cause a small amount of dysfunction in oxidative phosphorylation can actually promote lifespan.
Biogenesis is the creation of organelles. Increased biogenesis has been found to reduce aging by promoting healthy aerobic respiration. Mitochondria are always undergoing biogenesis. One gene that primarily controls this process is the peroxisome proliferator-activated receptor γ co-acticator 1α (PGC-1α). The expression and post-translational modification of this gene induces nuclear respiratory factors which are partially responsible for nuclear-encoding mitochondrial proteins. Biogenesis is especially important in skeletal muscle cells in which great amounts of energy are used in a single period.
Research done since the review publication
Since the Hallmarks of Aging paper  created in 2013, there have been multiple scientific research studies that further contribute to the understanding of this hallmark. Research done by Scialò et al. (2016) has found that ROS, originally thought to increase aging, actually has health benefits. The intentional increase of ROS can promote the extension of life through the reverse electron transport in the respiratory complex 1 of the ETC. This result is found in the model organism Drosophila melanogaster. This effect is seen only when there is an increase in this specific type of ROS. They can activate ROS-dependent pathways that help protect against damage and contain repair mechanisms. These pathways are thought to be how ROS contributes to longevity.
An additional study performed on Drosophila melanogaster studied the knockdown of mitochondrial ATP synthase subunit d (ATPsyn-d). This knockdown has demonstrated the ability to promote longevity. It is also shown that it is also associated with improved protein homeostasis and increased resistance to oxidative stress. It is suggested that it selectively alleviates oxidative damage from the mitochondria to maintain homeostasis in the cell and can vary in response based on acute or chronic stress and diet. The knockdown longevity is highly influenced by a high protein low carbohydrate diet. These effects were not seen in male Drosophila, which could be due to gender differences in physiology and nutrient uptake.
Another study done by Fleenor et al. (2013) showed that the spice, curcumin, has been found to reduce oxidative stress, improve vascular function, and increase lifespan. Curcumin comes from the plant Curcuma longa. This plant works by creating levels of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD) which reduces oxidative stress. Cucumin was also found to scavenge for free radials, another potential cause of aging.
The lifespan of Caenorhabditis elegans has been shown to increase when parts of the ETC have been diminished in activity. Mishur et al. (2016) created a specific mitochondrial mutant (Mit) that demonstrates these effects. It is seen that the effects of longevity on Mit mutants requires hypoxia-inducible factor-1 (HIF-1). HIF-1 is a protein with α and β subunits that helps with transcription in hypoxic conditions. Without this protein all effects are abolished even under normal respiration conditions. α-ketoglutarates are found to regulate HIF-1 and can accumulate in Mit mutants. These metabolites that accumulate after mitochondrial dysfunction directly affect the lifespan of the cell by increasing HIF-1.
More research has also been done on the effects of mitohormesis and ROS in the mitochondria using the drug, Metformin. Through the process of mithormesis, metformin increases the amount of ROS. This produced a decrease in the affect of aging in Caenorhabditis elegans, Rattus norvegicus, and Mus musculus. The affect on lifespan mimics a caloric restriciton model. Metformin inhibits complex 1 of the ETC causing electron flow to slow down and breathing rate to increase. After ROS are created they can go on to form hydrogen peroxide. Peroxiredoxins help alleviate the harsh affects of hydrogen peroxide, by scavenging for these molecules.
Exercise has also been found to have a profound affect on mitochondrial health. A study conducted on rats observed immobile young and old rats as well as active rats and looked at their PGC-1α mRNA and nuclear PGC-1α protein, This mRNA and nuclear protein are used to help stimulate mitochondrial DNA replication and transcription, which helps provide the mitochondria will the tools it needs for oxidative phosphorylation. Low levels of these were cognisant of age in the old immobile rats however some of these effects were reveresed in the old active rats demonstrating the endurance exercise can reverse or slow the affects of aging in skeletal muscle.
In the Hallmarks of aging review, the authors state that cellular senescence is brought upon by telomere shortening, non-telomere associated DNA damage and the deactivation of the INK4/ARF inhibitor. These means of causing cellular senescence occur as one ages. Due to the build-up of senescent cells increasing with age it has been hypothesized that cellular senescence is related to aging as the accumulation of the senescent cells may exasperate DNA damage by secreting proinflammatory secretome. The INK4a/ARF locus encodes for the p16INK4a and p53 pathways. These pathways induce senescence and have been linked physiological aging. The authors conclude that cellular senescence is necessary to cells as it halts the division of damaged cells however the accumulation of these senescent cells becomes toxic and accelerates aging.
Research done since the review publication
Much research has been done in the study of cellular senescence since the publication of the Hallmarks of Aging review in 2013. Such research has indicated that cellular senescence may be the driving force behind aging. In a review article titled "Cellular Senescence as the Causal Nexus of Aging" the authors explain that the reason aging is a gradual process because it requires the buildup of senescent cells to show signs of aging. They also indicate that there are various types of damage and stress that can induce cellular senescence. Other research suggests that cellular senescence is the leading cause for age related lung diseases in the elderly. Current research has shown that silencing small GTPase DIRAS3 can affect and induce cellular senescence in adipose stromal/progenitor cells and how this could be a way to control and prevent obesity and thereby extending lifespan. Researchers have also found that MicroRNA-29 can induce cellular senescence in aging muscles and increase the cell arrest proteins like p53, as mentioned in the Hallmarks of Aging review. Interestingly resveratrol has also been recently studied as a way to induce cellular senescence in cancer cells and how the effects of RSV could potentially be used as a form of cancer treatment. Whilst many avenues of cellular senescence is being researched, many researchers can agree that it is important to understand cellular senescence and it role on aging to potentially find ways to extend lifespan.
Stem Cell Exhaustion
Stem cells are undifferentiated cells, meaning that they do not have a specific function. Stem cells are important in maintaining tissue and organ function because as specific cells are damaged stem cells become specialized and replace the worn-out cells in the organism. Over time DNA damage, caused by mistakes, in the process of DNA replication, causes stem cells to become exhausted and cease replication. This inability to replicate and form new specialized cells causes damage to organs and tissues because there are no new cells to replace the damaged or worn down cells in the organism. This damage and wearing down of tissues and organs lead to aging in the organism.
Research done since the review publication
Since 2013, when the "Hallmarks of Aging," there has been major research into exactly what stem cells effect and how the body, as a whole, responds. This research has focused on such topics as classic telomere length, shown in a 2015 study "Telomere Dysfunction Causes Alveolar Stem Cell Failure." Other studies have shed light onto processes that were previously thought to be unaffected such as looking at DNA methylation and different pathways that hadn't been explored. These studies include "Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging" and "Loss of aryl hydrocarbon receptor promotes gene changes associated with premature hematopoietic stem cell exhaustion and development of a myeloproliferative disorder in aging mice." These studies show a change in research conducted since the 2013 "Hallmarks of Aging" was published.
The article “Proliferation-Dependent Alterations of the DNA Methylation Landscape Underlie Hematopoietic Stem Cell Aging" published by Beerman is significant because this article tackles many different hypothesizes that were discussed in the 2013 article but not researched. One of the major findings from this article is that telomere length is independent of stem cell DNA methylation and age-related problems. This is radically different than previous research that said there was a link between the two. This article also looked at global DNA methylation and debunked a theory that DNA methylation increased over time when, according to this study, DNA methylation was consistent throughout aging.
Other studies that are more focused and not as wide-reaching have discovered specific pathways that affect the mechanisms in which stem cell lose their function and viability. One such study was "The Polycomb Group Gene Ezh2 Prevents Hematopoietic Stem Cell Exhaustion" This study focused on the reasons for stem cell loss of function with regards to aging wild types. They discovered the Enhancer of zeste homolog 2 (Ezh2) which was found to be the most expressed transcript in hematopoietic cells. They then overexpressed Ezh2 and found that they could overcome cellular senescence and they conserved repopulating potential, of the stem cells, long after the wild-type cells had been exhausted. They concluded that this Ezh2 helped with stabilization that helped conserve the stem cells from stress related to aging.
A study published in January 2017 was "Increased Arf/p53 activity in stem cells, aging and cancer" published in the journal Aging Cell. This study focused on the Arf/p53 pathway and how its role in cancer suppression is affected by quantity and age. They found that increasing p53 delays stem cell exhaustion and decline of homeostatic tissues. However, they also observed that if p53 is being constantly activated then the pathway accelerates the aging process of stem cells which reduces tissue regeneration and replicative regulation.
A new study, "RNA Editing-Dependent Epitranscriptome Diversity In Cancer Stem Cells", found that cancer stem cells (CSCs) can regenerate all the parts of the tumor and unless destroyed can regenerate all of the affected cells. This study aimed to examine what exactly contributed to CSCs ability to proliferate and their maintenance. This study found that the more damage, caused by DNA methylation and incorrect RNA editing and splicing allowed for these CSCs to form and from them allowed all facets of the tumor to proliferate.
Altered Intercellular Communication
As aging progresses, changes or alternations in intercellular communication occur. These intercellular communications consist mostly of three types. The first type is neuronal which consists of communication that only happens between the cells of the nervous system, specifically the neurons. The second type is neuroendocrine which consists of communication that happens between the nervous system neurons and the endocrine system's hormones. Last is endocrine which consists of communication that only happens between the cells of the endocrine system. Any of these three types of communication can be affected in the process of aging. Research has shown that altered intercellular communication due to aging leads to increased inflammation, reduced neurogenesis and decline in efficient autophagy. 
Research done since the review publication
Previous research done by Zhang et al. in 2013 found that the inhibition of IκB kinase-β, IKK-β, and nuclear factor κB, NF-κB, in the hypothalamus caused an increase in gonadotropin-releasing hormone, GnRH, which led to adult neurogenesis. Thus, Zhang et al. were cited in “The Hallmarks of Aging” under altered intercellular communication for their findings. Later research after 2013 also agreed with Zhang et al. that IKK-β and NF-κB is an integral part of aging and longevity.  In recent years, research in altered intercellular communication in regards to aging and longevity has shifted from studying the neuronal network into examining how the circadian rhythm and cycle affects the hippocampus and neurogenesis.
However, circadian rhythm and cycle was not the only aspect being studied. Research was also conducted on various receptors, proteins, and hormones that had a connection to the effects of altered intercellular communication to see if they were the cause, effect, or otherwise. In addition, these receptors, proteins, and hormones were examined and manipulated extensively to see if they were able to improve the effect of altered interceullar communication or not.   In early 2017, Lacoste et al. proposed the malfunction or impairment of the circadian clock as the tenth hallmark of aging. They suggested that aging, the circadian clock and cellular oxidative stress are interrelated, because they propose decrease efficiency of the antioxidant defense system with age. However, some of their findings about the levels of lipoperoxidation, LPO, and glutathione, GSH, as a factor of measuring oxidative stress as a person ages does not agree with earlier research. The reason behind this is still hugely unknown until further research has been conducted.
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