Course : PGPathshala-Biophysics
Paper 11 : Cellular and Molecular Biophysics Module 7 : Aging and Longevity
Content Writer : Ashikh Seethy, AIIMS, New Delhi Introduction:
Staying young and preventing death has been a prime priority of humans since time immemorial. Indeed this was the driving force for ancient sciences like alchemy and the concept of “elixir of life” has been mentioned in various ancient scriptures of Greeks and Indians as “Ambrosia” and “Amrit” respectively.
Despite centuries of research, we are not able to prevent death. But science has achieved major breakthroughs that has helped in prolonging the human life span. We also now know about the various mechanisms of ageing and senescence. In this module, various aspects of ageing and longevity will be discussed from a biochemical perspective.
Objectives:
At the end of this module, you should be able to:
1. Explain Hayfick’s limit
2. Explain various biochemical mechanisms of ageing 3. Explain the mechanism of telomerase
4. List various causes of premature ageing 5. List biomarkers of aging
1. Why do we age?
The simplest answer is: we age because our cells age. In the earlier half of 20th century, it was believed that normal vertebrate cells have a limitless replication potential. Nobel laureate Alex Carrel was an advocate of this hypothesis. In 1960s, an American Anatomist, Leonard Hayflick conducted a series of experiments to disprove this hypothesis. He maintained in vitro cultures of normal human male fibroblasts that had undergone large number of cell divisions (eg. 49 times), along with normal human female fibroblasts that were relatively “young” (eg. 13 cell divisions). When the cultures were examined after a time duration required for 17 cell divisions or so, it was observed that only female fibroblasts were present. This concluded that fibroblast cells failed to survive after a certain number of cell divisions. (1) The same the case with various adult and fetal cell types from different tissues all of which failed to divide more than 40-60 times. This limited potential for somatic cell to undergo mitosis is known by the term “Hayflick’s limit”. This limited dividing capacity of the cell is responsible for cellular aging and eventually human aging. We will explore various mechanisms that are responsible for Hayflick’s limit subsequently.
2. Mechanisms of Ageing
Once it was established that somatic cells have a limited replicative potential and they die after a fixed number of cell divisions, focus was shifted to decipher the underlying mechanisms behind this.
It was akin to a revisit to the age old quest of finding the elixir of life. Intriguing mechanisms that contributed to cellular senescence were revealed- some of them are explained below:
2.1 Shortening of telomeres:
Telomeres and end-replication problem
Telomeres are repetitive sequences at the ends of linear chromosomes. A chromosome is nothing but a condensed molecule of DNA. Because of the linear nature of the eukaryotic chromosomes, there
occurs a peculiar phenomenon in these chromosomes during DNA replication. This is known as end- replication problem.
DNA polymerases can add a nucleotide only to a pre-existing nucleotide polymer. Thus, an RNA primer is an essential prerequisite for the DNA polymerases to initiate polymerisation during DNA replication. These RNA primers are eventually degraded by enzymes like RNase H1 and FEN1, and are replaced with deoxy-ribonucleotides by DNA polymerase δ.
The following figures summarize the end-replication problem:
During replication, RNA primers are synthesized by the primase activity of DNA polymerase α. The figure shows the telomeric end of a chromosome.
The leading and lagging strands are synthesised by DNA polymerase δ. Note that DNA polymerase δ can add nucleotides only to a pre-existing nucleotide oligomer. Leading strand is synthesized in a continuous manner while the lagging strand is synthesized discontinuously in the form of Okazaki fragments.
RNA primers are then degraded by enzymes like RNase H1 and FEN1.
DNA polymerase δ now fills these gaps due to its polymerase activity, and the nicks are sealed by DNA ligase. Since there is no pre-existing nucleotide oligomer at the 5’ end of the newly synthesized leading strand, DNA polymerases cannot fill the gap here. This leaves a 3’ overhang at the end of the chromosome with loss of DNA at the 5’ end. The same happens at the 5’ end of the newly synthesized lagging strand also, due to the same mechanism.
With progressive cell divisions, the telomeric ends suffer attrition and this leads to cell cycle arrest.
Telomerase
Certain eukaryotic cells like germ cells and stem cells express an enzyme called telomerase, to counter this end replication problem. Telomerase is a reverse transcriptase, i.e., it is an RNA dependent DNA polymerase. The intriguing feature of telomerase is that the RNA template which is required for DNA synthesis is present with the enzyme itself. Thus, telomerase is a ribonucleoprotein.
How does telomerase act? Human telomeres are rich in the repeating AGGGTT sequence in the 3’
end. The RNA template present in the telomerase is complementary to this sequence and extends beyond the AGGGTT unit. Thus it can bind to the 3’ end of the chromosome and synthesize new DNA at the 3’ end, using its own RNA as template.
Telomerase then translocates to the 3’ end and continues this process, extending the 3’ end of the chromosome. The purpose of this extension of 3’ end is that a new RNA primer can bind to this strand.
DNA polymerase now adds nucleotides to the newly bound RNA primer. Thus the 5’ end of the newly synthesized strand is restored. As the ends of chromosome has now been sufficiently extended, removal of the RNA primer by ribonucleases won’t affect chromosome integrity, even though it leads to a 3’ overhang. This 3’ overhang usually invades the intact DNA duples to form a T- loop which is further stabilized by proteins like TRF1 and TRF2. The T-loop protects the chromosome ends from nucleases.
Telomerase is either absent or is expressed at very low levels in somatic cells. Studies in mice have shown that absence of telomerase in the animal as a whole lead to accelerated aging manifested by stem cell depletion, tissue atrophy and organ failure. Reactivation of telomerase in such animals eliminated the degenerative phenotypes and restored near normal phenotypes.(2) If this is the case,
can telomerase be used to prevent aging of somatic cells and thus aging in the whole organism? At this point of time, the answer is no. This is because of the fact that apart from germ cells and stem cells, cancer cells also consistently express telomerase. Telomerase expression is responsible for replicative immortality, which is one of the hallmarks of cancer.(3)
Thus cells overexpressing telomerase are prone for malignant transformation. Though studies have shown that the lifespan of individuals with increased telomerase activity are longer (4) and there are reports of telomerase administration to humans with the aim of increasing longevity, currently there are no peer-reviewed studies on humans are available about any telomerase trials to prevent aging.
Though telomerase is not used in anti-aging therapy, targeting telomerase is a potential strategy to curb proliferation of cancer cells. Imetelstat, an inhibitor of telomerase is in phase 2 and 3 trials for treatment of premalignant haematological conditions like myeloproliferative neoplasms and myelodysplastic syndromes.(5) Immunotherapy targeting telomerase has also been explored, albeit without success.(6)
Laboratory applications:
Telomerase is used as a biomarker for diagnosis and prognosis in certain types of cancer.
hTERT (human telomerase reverse transcriptase) is used in immortalisation of primary cells isolated from tissues. This helps in establishment of an immortalized cell lines.
Points to ponder:
1. The end replication problem is because of the requirement of an RNA primer for initiation of replication instead of a DNA primer. What is the probable advantage of an RNA primer over a DNA primer?
2. Telomerase is an enzyme that contains RNA and protein. Can you call it a ribozyme?
3. Is there any end-replication problem in prokaryotes?
4. Apart from the usage of hTERT, what are the other strategies to immortalize cell lines?
2.2 DNA and Protein Damage:
Our cells are exposed to numerous physical and chemical factors that are present in the internal and external environments. Over the period of time, exposure to certain unfavourable physical and chemical agents is detrimental, as they cause alterations in the DNA and proteins of the cell.
Free radicals:
Free radicals refer to those chemical species with an unpaired electron in its outer orbital. A closely related but different species is reactive oxygen species (ROS) and reactive nitrogen species (RNS).
ROS includes superoxide radical, hydroxyl radical, hydrogen peroxide etc while RNS includes peroxynitrite radical, nitric oxide etc. As you can see in the figure, not all ROS (and RNS) contain an unpaired electron in their outer orbital. Thus all ROS and RNS are not free radicals. However, even those ROS and RNS that are not free radicals can generate free radicals and are deleterious. These molecules are highly active due to the unpaired electron and can readily react with a variety of molecules in a cell including nucleic acids, lipids and various proteins.
Biological oxidation is a major source of free radical generation. NADH and FADH2 donates their electrons to the complexes I and II respectively of the electron transport chain located in the inner mitochondrial membrane. These electrons are transported along complex III to complex IV, where oxygen is the final electron acceptor, and subsequently converted to water. The efficiency of this process is diminished in older mitochondria, which leads to electron leaks at various complexes.
Normally a single molecule of oxygen accepts 4 electrons and 4 protons within the complex IV to generate two molecules of water.
O2 + 4H+ + 4e- 2H2O
Due to mitochondrial electron leakage, the oxygen may accept 1, 2 or 3 electrons leading to the formation of superoxide, peroxide and hydroxyl ions respectively, the latter being the most potent free radical.
Apart from this, free radicals are also generated during metabolic processes like prostaglandin synthesis, phagocytosis (NADPH oxidase enzyme generates free radicals that are toxic to microbes), inflammation, xenobiotic metabolism etc. Transition metals like iron and copper can also catalyse free radical generation by Fenton reaction
H2O2 + Fe2+ Fe3+ + OH. + OH- Free radicals can cause cell injury and aging by:
-DNA double strand breaks, single strand breaks, formation of DNA adducts etc. The latter can be hazardous because certain oxidised bases and nucleotide adducts are identified different from their parent bases by the DNA polymerases. This leads introduction of mutations in the daughter strand during replication. In presence of free radicals, the DNA repair mechanisms are overwhelmed and alterations in DNA can lead to cell cycle arrest and senescence. Studies in hematopoetic stem cells (HSCs) revealed that aging HSCs have an altered phenotype compared to young HSCs and the latter has a higher rate of differentiation and homing efficiency when transplanted. This could be due to the cumulative effect of mutations that has occurred to the HSCs as they have aged.(7)
-Free radical induced damage can affect protein function and can enhance protein degradation.
Protein adducts can also lead to abnormal protein folding. When unfolded proteins accumulate, the cell resorts to a salvage mechanism known as unfolded protein response.
Studies have shown that mice with low expression of chaperones age prematurely and overexpression of chaperones lead to increased longevity.(8) If all the other mechanisms fail, the cell will undergo translational arrest and senescence, or in extreme situations, apoptosis or autophagy.
- Membrane lipid peroxidation: Free radicals can react with lipid components of the biological membranes like PUFA and can cause widespread membrane damage. They also can oxidise lipid components of molecules like low density lipoprotein (LDL) to form oxidised LDL, which is highly atherogenic.
Ultraviolet radiations:
UV radiations of the wavelengths 200-280 nm (UVC) is the most harmful of the UV rays, but these are filtered by the ozone layer. UVB (280-320 nm) is biologically active but cannot penetrate superficial skin layers. Exposure to UVB can predispose to skin cancers. UVA (320-400 nm) can penetrate beyond superficial skin layers and is responsible for ageing of skin.
Exposure to harmful UV rays can cause direct DNA damage in the form of pyrimidine dimers and chromosomal instability. In prokaryotes pyrimidine dimers are corrected by an enzyme called DNA photolyase, but this enzyme is absent in eukaryotes. Eukaryotes rely mainly on nucleotide excision repair (NER) for the removal of pyrimidine dimers. Patients with a clinical condition called xeroderma pigmentosa have defective NER and they are prone to skin cancer and premature aging.
The alternative pathway for repair of pyrimidine dimers is a specific form of DNA replication known as translesion DNA synthesis (TLS), catalysed by DNA polymerase η, which is an error prone DNA polymerase. Defect in DNA polymerase η leads to a similar clinical picture as in xeroderma pigmentosa.
UV rays can also lead to generation of free radicals and DNA and protein damage, adding salt to the sore.
Points to ponder:
1. We have discussed the adverse effects of free radicals. But do they perform any normal function?
2. Is it likely that oxygen is actually a slow poison and it just takes 75–100 years to kill us?
2.3 Nutrient Sensing:
The adage goes like “we are what we eat”. Surprisingly this holds to some extent as far aging is concerned. Evolution has designed our bodies to cope up with nutrient deficiencies, as food was a scarce resource to the early hominids. Studies in mice have shown that calorie restriction can increase lifespan by up to 50% in controlled conditions. Sensing of a particular nutrient can be mediated directly by the molecule binding to its putative sensor, or can be indirect due to some surrogate molecule downstream the nutrient metabolism. For instance, glucose is sensed by glucose transporter (GLUT2) and glucokinase, cholesterol by cholesterol-sensing protein SCAP (SREBP1 cleavage activating protein), lipid storage by adipokines like lectin and energy balance by AMP activated protein kinase and various enzymes that respond to altered ATP:AMP ratio.
Increase in carbohydrate and protein intake is associated with an enhanced signalling involving insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway. This leads to and increased generation of mTOR (mechanistic/ mammalian target of rapamycin). mTOR is associated with inhibition of autophagy, which is required for clearance of damaged and misfolded proteins. Inhibitor
of mTOR, rapamycin has been shown to relieve symptomatic effects of aging, probably by promoting autophagy.(9) Similar effects are postulated to be exhibited by an inhibitor of IIS pathway, metformin, a widely used oral hypoglycemic agent.
Sirtuins are protein NAD dependent deacetylators (histone deaetylators) that form a link between nutrition status and gene expression. Sirtuins 1 and 2 are associated with an increased lifespan in animal studies, and expression of these sirtuins are increased by calorie restriction.(10) Deacetylation by sirtuins has been shown to activate proteins like isocitrate dehydrogenase, which can lead to NADPH production and activation of NADPH dependent anti-oxidant systems. Resveratrol present in red wine is a sirtuin activator and a potential candidate for anti-aging therapy.
2.4 Epigenetics and Aging:
Epigenetics refers to the heritable changes in the genome without any change in the sequence of DNA. Epigenetic mechanisms play a prime role in regulation of gene expression. Apart from histone deacetylation discussed above, and other histone modifications, DNA methylation is a major epigenetic mechanism and has implications on aging.(11) 5-methyl cytosine (5-mC), an epigenetic modification, mostly exists in the CpG dinucleotide context-70-80% of CpGs are methylated. These are commonly found in the 5' end of the genes. The methyl group of 5-mC protrudes into the major groove of the DNA where it interferes with the binding of transcription factors.
Studies in aging hematopoietic stem cells have shown that expression of 1500 genes were increased and 1600 genes were repressed. Those that were induced included genes involved in inflammation, protein aggregation etc while those that were repressed were involved in protective mechanisms like maintenance of genomic integrity.(11) Aging is associated with an alteration in the CpG methylation- a process known as age related methylation drift, with alteration in the methylation patterns across promoters and heterochromatin. This could be responsible for altered gene expression associated with aging. Indeed caloric restriction has been shown to counter age related methylation drift.
3. Premature Aging Syndromes:
We have already seen that defective DNA repair (nucleotide excision repair and translesion DNA synthesis) leads to premature aging in xeroderma pigmetosa. Not surprisingly, a battery of conditions can lead to premature aging, most of them involving a defective DNA repair.
Disease Gene defective Pathway defective Function of the protein
Ataxia telangiectasia
ATM (Ataxia
telangiectasia mutated)
Double strand DNA break repair
Activation of p53 by phosphorylation to facilitate DNA repair.
Maintains stability of dsDNA strands during recombination Bloom
syndrome BLM Double strand
DNA break repair
BLM is a helicase that is involved in DNA
recombination and repair Fanconi
anemia
Multiple genes like FANCA, B, C, D2, E, F, I, L, M, D1/BRCA2
Double strand DNA break repair
The genes involved encode a protein complex that is
involved in homology directed DNA repair
Nijmegen breakage syndrome
NBN (Nibrin) Double strand DNA break repair
Nibrin is involved in end- processing of physiological and mutagenic DNA double-strand breaks
Werner
syndrome WRN Double strand
DNA break repair
WRN protein is a helicase similar to BLM that is involved in DNA recombination and repair
Hutchinson Gilford progeria syndrome (HGPS) is another well characterised premature aging syndrome. The protein defective is lamin, which is a component of the intermediate filaments that form the nuclear lamina. The defective lamin protein (also known as progerin) is a truncated from of lamin A precursor protein (prelamin A) that is farnesylated. Normally farnesylated portion of prelamin A is cleaved by proteases to from lamin A, but mutations lead to loss of the site at which the proteases act. Defarnesylated prelamin A accumulates in the nuclear membrane and disrupts normal cellular function. Inhibitor of farnesyltransferase, lonafarnib, has been found useful in ameliorating manifestations of this syndrome.(12)
4. Biomarkers of Aging:
Biological age of a person might be different from chronological age. Since biological age is a risk factor for various morbidities including cardiovascular diseases and cancer, biomarkers that can reflect biological age can have various clinical utilities. Further, these biomarkers can also be of use in assessing the effectiveness of any intervention that can prevent aging.(13)
Since aging is a complex procedure, there is no single ideal biomarker for aging. Various proposed markers include but not limited to:
1. Telomere length in leukocytes.(14) This can be measured by a technique known as Terminal Restriction Fragment (TRF) analysis. This assay is a modified southern blotting and utilizes restriction enzymes that does not cleave telomere sequences. Telomere specific probes are used after restriction digestion, electrophoresis and blotting of the DNA fragments to determine the telomere length.
2. Telomerase activity can be estimated using Telomere Repeat Amplification Protocol (TRAP).
Here telomerase from the sample is added to a telomerase substrate (an oligonucleotide molecule). Oligonucleotide specific primers are used to perform a PCR amplification. If telomerase activity was intact, an amplicon will be formed and vice versa.
3. Phosphorylated histone protein (H2AX) is a marker for DNA repair and can be detected using immunohistochemistry.
4. Age related changes in DNA methylation: Aging is associated with loss of methylation in heterochromatin regions, while promoter CpG islands are hypermethylated.
5. Non-coding RNAs: Certain miRNAs like mir-21 and long non-coding RNAs like meg3 were found to be associated with an increase in biological age.(15,16)
6. As mentioned previously, molecules related to nutrient sensing such as those related to insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS) pathway and mTOR (mammalian target of rapamycin) pathway can act as biomarkers of aging.
7. Advanced glycation end-products (AGE) are formed due to non-enzymatic addition of carbohydrates to proteins, lipids and nucleic acids. Accumulation of AGE is associated with age related disorders and cellular dysfunction. AGE can be measured by specific assays or using chromatographic techniques like HPLC or gas chromatography.
5. Evolutionary Perspective of Aging
Aging is associated with a progressive deterioration of various bodily functions. The plight of ageing is described by William Shakespeare in his play As You Like It:
“And so, from hour to hour, we ripe and ripe, And then, from hour to hour, we rot and rot;
And thereby hangs a tale”
The end of life care has significantly improved since the Elizabethan era, still the question remains- why should we age?
Nothing in biology makes sense except in the light of evolution.(17) Evolution selects traits that contribute to the fitness and survival of the species, so the fact that organisms age and wither away seems to be logical fallacy; but this is not true. A prolonged lifespan may be of benefit to an individual, but the rules change when it is applied to a species as a whole. A limited lifespan also limits draining of resources getting diverted to that fraction of population who are not involved in reproduction. The purpose of evolution is the maintenance of genes that are beneficial for survival and this is achieved during the reproductive years, when the genetic material is transferred to the offspring. So traits that prolong life beyond the reproductive age will not have any positive selection pressure.
6. Summary:
1. Somatic cells have limited replicative potential (Hayfick’s limit) and shortening of telomeres is mainly responsible for this.
2. Stem cells, germ cells and cancer cells exhibit telomerase, which maintains telomere length.
3. Free radicals can induce damage to DNA, proteins and membranes. Anti-oxidants have a favourable role in delaying aging.
4. Excess calories and nutrients can alter nutrient sensing mechanisms to accelerate aging.
5. Aging is associated with epigenetic alterations and altered gene expression.
6. DNA repair mechanisms are affected in many premature aging syndromes.
7. Different biomarkers to identify biological age have been proposed.
