Ageing Biology
Content
SCRA.18 AB.01 AGEING BIOLOGY 01 – Introduction to the Biology of Ageing The Ageing Biology focuses on the ageing process and ageing-related diseases. Principles from the Stem Cell Biology and Regeneration Lectures will be used. It is important to understand the effects of ageing on different levels: the molecular, cellular, tissue and organ. Key objectives - Appreciate the costs of our ageing population and the need for research if we are to enable healthy ageing - Describe the characteristics and a possible definition of ageing - Know that there are several segmental progeroid syndromes that could give us clues as to human ageing - Describe the advantages of invertebrate model organisms and what they have told us about possible mechanisms of ageing including SIRTs, TOR and IGF signalling (more in later Lectures) The worldwide populating is ageing and people are living longer. It has been predicted that by 2025 1.2 billion people will be over 60 years old and many more people will be over 80 and above. This will mean that more people will be living with multiple chronic conditions.
Defining Ageing There are many changes that occur as an organism ages: accumulation of nuclear and mitochondrial DNA damage due to oxidative stress, gradual immunodeficiency leading to decreased immunity to infections, organs lose their ability to maintain homeostasis because signalling pathways are damaged and stochastic accumulation of tissue damage.
PROGEROID SYNDROMES Progeroid syndromes (PS) are a group of rare genetic disorders that mimic physiological ageing, making affected individuals appear to be older than they are – accelerated ageing. These syndromes can affect many organs – segmental or one organ – unimodal. Segmental progeroid syndromes: > Werner syndrome > Ataxia telangiectasia (AT) > Dyskeratosis congenital (DC) > Hutchinson-Gilford progeria syndrome (HGPS) Unimodal progeroid syndromes: > Familial Alzheimer’s disease > Familial Parkinson’s disease
I. Werner syndrome WS is caused by an autosomal recessive mutation and affects 1/100,000 people. Sufferers develop normally until puberty when they stop growing and show premature ageing. Symptoms: cataracts, grey hair, osteoporosis, cancer, atherosclerosis, poor glucose regulation, immunodeficiency, osteoporosis, skin atrophy, myocardial infarction, brain symptoms are rare. The median age of death is 47-48 years old caused by myocardial infarction and cancer. Mutations in the WRN gene lead to proteins without a nuclear localisation signal (NLS). The protein is a RecQ helicase enzyme that unwinds DNA and is involved in genome maintenance and stability. Wrn proteins are involved in DNA repair, recombination and telomere maintenance. The helicase unwinds the DNA in a 3’-5’ exonuclease activity. Werner cells in vitro undergo telomere shortening, fewer divisions, premature senescence and incorrect recombination events – translocations and deletions. Cells treated with H2O2 will initiate DNA damage signals and trigger cell senescence as Werner cells. Werner cells have different responses to oxidative stress.
II. Ataxia telangiectasia (AT) AT is caused by a rare autosomal recessive mutation in the ATM gene. The ATM protein is a kinase that phosphorylates proteins involved in DNA damage signalling, DNA repair and telomere maintenance. AT cells show telomere shortening, genome instability and premature senescence. It is a progressive cerebellar degeneration – the Purkinje cells die leading to ataxia. Telangiectasia – tiny red veins develops and the skin develops pigment abnormalities and hair greying. Melanocytes cannot cope with accumulating DNA damage and so die, and so not enough melanin is produced. Other symptoms include immunodeficiency and a wide range of aggressive malignant tumours such as leukaemia and lymph gland cancer. Ataxia is the characteristic symptom – uncontrolled muscle movement. III. Dyskeratosis congenital (DC) DC is a rare, inherited, usually male disease that causes the degeneration of mucous membranes, teeth, nails and skin pigmentation. Patients can develop cancers, grey hair, and osteoporosis and have bone marrow dysfunction. 6 DC genes have been identified that are all involved in telomere maintenance (telomerase components or telomere cap). The genome becomes unstable when these genes are mutated. Agarwal et al, 2010 in Nature Fibroblasts from DC patients were transduced with a cocktail of transcription factors (Oct5, Klf4, Sox2 and c-Myc) to induce them into a pluripotent state. The iPS cells were found to have longer telomeres showing that the upregulation of the telomerase RNA component (TERC) is part of pluripotent state. When these cells differentiate, TERC levels fall and telomeres become shorter. Several telomerase components are targeted by pluripotency-associated transcription factors.
IV. Hutchinson-Gilford progeria syndrome (HGPS) HGPS is a caused by a dominant deletion of 50 amino acids in the lamin A, a protein that forms part of the lamin mesh underneath the nuclear envelope. The protein is truncated and so it is incorrectly spliced and processed – it stays farnesylated. Farnesyl is a 15 carbon lipid molecule. This mutant ‘progerin’ protein accumulates in cells. It affects the nuclear envelope and chromatin because lamins have both structural and signalling roles involving the Wnt pathway. The incorrect interaction between the progerin and genome leads to DNA damage and senescence. Does the lamin directly interact with chromatin and influence gene expression e.g. of Notch effectors? HGPS cells in culture have a reduced lifespan and display irregular nuclear phenotype: chromatin organisation is altered there is less heterochromatin, blebbing and binucleated cells. Telomere lengths are reduced and telomeres aggregate. The response to the DNA damage is chronic as the progerin lamin mesh is not changed. Progerin signals to p53 and Rb pathways that lead to cell cycle arrest and early cell senescence. Non-HGPS patients will produce more progerin as they age, Sufferers develop symptoms before the age of two. Sufferers have very thin skin; they lose subcutaneous fat, alopecia, stiff joints, and osteoporosis and heart degeneration. The mean age of survival is 13 years usually caused by a heart attack or stroke.
Hernandez et al., Dev. Cell, 2010 The defects are associated with inhibition of canonical Wnt signalling, due to reduced nuclear localisation and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. LEF-1/TCF transcription factors mediate a nuclear response to Wnt signals by interacting with beta-catenin. Defective Wnt signalling, affecting ECM synthesis may be critical to the aetiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. This might explain why progeria patients have skeletal problems and thinning/apoptotic blood vessels. This could also be the reason why old blood vessels can thin. HSPG models The knockdown of Zebrafish lamin A/C induces laminopathies associated with muscular dystrophy and craniofacial abnormalities. 2013 clinical trial Lonafarnib is a small drug that has been trialled to help patients with HGPS. It is an inhibitor of farnesyltransferase (FTase). This enzyme adds the farnesyl lipid moiety to lamin A proteins. 25 children with HGPS participated in the open-label trial. There was body weight gain, the arteries appeared to be less stiff (as judged via measurements of carotid-femoral pulse-wave velocity), and bone rigidity measurements and bone mineral density were improved relative to before drug treatment. DNA damage Progeroid syndromes have one thing in common – they lead to genomic instability. They suggest that genomic instability is a key factor in ageing. Instability e.g. DNA damage, telomere integrity, etc. is dangerous because it leads to cancer, senescence and cell death. Stress responses will be initiated to try and repair the damage. Cells can only cope with a certain amount of DNA damage and will eventually apoptose or enter quiescent. However do progeria syndromes really mimic aspects of ageing? Evolutionary aspect It may be that there are mechanisms that have evolved over time to ensure survival and maintenance of reproductive organs until reproductive age. Past this age, there is no selective advantage to keep these mechanisms and this may explain ageing and the degeneration of tissues and organs. However there may be selective advantage for older people to look after their grandchildren freeing up energy and time to produce more offspring. STUDYING AGEING The ageing process is gradual and heterogeneous. The ageing rate is affected by both environmental and genetic factors. There are no biomarkers that can be used to study ageing and the changes in brings. Model organisms are used but there are advantages and disadvantages to using them. Ageing is very expensive and difficult to study, especially in humans. Subjects will need to be followed throughout their lifetime. This is expensive even in animal models which live for a long time too: Yeast (Saccharomyces cerevisiae) – 3 days, 25-35 generations Caenorhabditis elegans – 12-20 days Flies (Drosophila) – 50-80 days Rodents – less used, 18 months-4 years Humans – up to 80-100 years
Therefore there are very few cohort studies. Cohort studies are difficult to standardise because there are so many variables to control e.g. different genes and environment of each participant and different ageing rates of different organs. Also different participants will have different ageing dynamics, all these variables lead to noise. These model organisms are cheaper and have a shorter timescale. Furthermore the variables are easier to control in a laboratory environment. Observed changes in physiology may not be harmful and preventing them in experiments may be detrimental to the organisms’ health.
Ageing involved the gradual accumulation of changes on different levels at different rates because the rate of ageing is not always the same in any individual. Each tissue will respond to ageing in its own way: muscle and fat tissues undergo atrophy; wound healing and the immune response become compromised. Ageing is a major risk factor for many different chronic and fatal diseases e.g. cardiovascular diseases, arthritis, Type II diabetes, cancers… Ageing is very economically costly as health, transport and social infrastructure has to be specially modified to cope with the unique demands old age brings.
MODEL ORGANISMS Budding yeast Yeast is a simple organism and has been a staple model organism. Its small genome has already been sequenced. They are quick and cheap to culture, easily genetically manipulated e.g. knockout strains, and analysed. There are many gene orthologues in humans, so any findings in yeast may be translatable to humans. Yeast have a finite replicative capacity = ‘replicative lifespan’ (RLS) and they also have a limit to how long they can survive in a non-dividing state = ‘chronological lifespan’ (CLS). The molecular basis of RLS and CLS can be studied. Different yeast strains can be cultured in defined conditions. Old yeast display many physiological changes. They are much larger and their cell cycle and protein synthesis is slower when compared to younger yeast cells. The cell surface appears ‘loose and wrinkled’. Old yeast also have bud scars. Old cells express some apoptotic markers. Yeast are an important model organism because their ageing biology has been conserved in humans, showing that there are fundamental features of ageing present in most organisms. Important biomolecules SIRTUINS – silent information regulator 2 family of protein deacetylases/Sir2 proteins Sir2 proteins are an ancient and highly conserved family of proteins found in all forms of life and display a structure and catalytic function that have been maintained from bacteria to humans. Sir2 proteins are NAD-dependent deacetylases that were originally discovered to regulate longevity in budding yeast by Kaeberlein et al, 1999 and Imai et al, 2000. NAD is an electron carrier in the electron transfer chain. Sir2 proteins are involved in gene silencing by chromatin deacetylation. They also act as nutrient sensors and regulate many aspects of physiology. Sir2 proteins set the rates of cell division, metabolism and protein synthesis. Deletions of Sir2 have shown to shorten the replicative lifespan of yeast. Overexpression increases the RLS. Nutrient sensing and ageing (Guarente, 2013 Review) Caloric restriction without malnutrition has been proved to increase lifespan in mammals and yeast e.g. reduced glucose and/or amino acids broth for the yeast. During times of plenty, cells will have high metabolism and produce less NAD+ (more NADH and H+) as they use up NADH to produce ATP for biosynthesis and growth. Sir2 is inactive as more genes are highly active (increased protein synthesis) and the reproductive activity is high therefore the RLS is normal. Conversely during periods of low calorie input, metabolism slows down and there is more NAD+ in cells. Sir2 is active because it silences genes reduce metabolism and reproduction activity. The RLS is longer than normal.
These observations present an interesting dichotomy. Cells have a ‘choice’: to reproduce or live longer. These effects have been demonstrated in mammals, rodents, C. elegans, Drosophila and yeast. It is important to note that there have been independent groups both showing that Sir2 proteins do and do not extend life. This may be caused by differing genetic background. However the consensus is that sirtuins play a role in extending life. Sirtuins systematically redirect mammalian physiology in response to diet. Sir2 proteins reduce metabolic activity and maintain it at the baseline level and increase glycolysis efficiency.
TOR KINASES – Dibble & Manning, 2013, Nature Review Target of rapamycin (TOR) is a serine/threonine kinase that is a master regulator of metabolism and growth. It is a convergence point of a vast signalling network to sense changes in extracellular and intracellular nutrients, and responds accordingly by regulating metabolism, growth and proliferation. TOR proteins are nutrient responsive and so have increased activity when nutrients are high. A decrease in TOR activity increases RLS and CLS. Many physiological signals affect the activation status of TOR. TOR proteins receive various signals and integrate them into output signals that lead to appropriate downstream effects. TOR is involved in various signalling pathways that may be involved in the ageing process. >> When TOR activity is reduced due to nutrient starvation, stress response factors enter nucleus, leading to resistance of oxidative and temperature stress. Experimentally, the addition of rapamycin will lead to these downstream effects. >> In mitochondrion–to-nucleus signalling, in response to mitochondria dysfunction TOR can decrease RLS in yeast. >> TOR represses autophagy and this can reduce yeast CLS. Autophagy is a starvation response, where cells digest non-essential organelles as an energy source. >> TOR is involved in coordinating metabolic changes in response to nutrient changes e.g. in yeast: a change to respiration from fermentation when cellular glucose levels falls. This may increase RLS and/or CLS. >> TOR promotes transcription of ribosomal proteins (RPs) and rRNA processing factors. If a cell is starving or TOR is inhibited transcription is greatly reduced and translation is impaired. This leads to decreased ribosome biogenesis and protein translation. Conclusions from yeast studies Yeast replicative lifespan and/or chronologic lifespan can be extended via Sir2 overexpression, TOR-inhibition, Ch9/Akt (later) or dietary restriction. The genetic mechanisms behind these processes are conserved in worms, and flies and mammals. However there are limitations to using yeast as model organisms for humans. It is not known just how much of yeast biology is conserved and therefore applicable to humans. Yeast have different DNA modifications e.g. no methylation, no telomere shortening with age.
C. elegans as a model organism Much is known about the nematode worm. The 1mm long adult has exactly 959 cells whose exact lineage from zygote to somatic cells has been mapped. The nematodes are very short-lived and easily cultured, and the genome has been sequenced too and there are many genetic resources available. Therefore data is obtained very quickly. The nematode shares orthologues with other species but lacks some crucial families e.g. Shh. Nonetheless it undergoes functional and ageing senescence as mammals and humans do. Old worms have muscle atrophy (sarcopenia), reduced skin elasticity and vulnerability to infections. If there is enough food, the development of egg to adult occurs in 3-4 days. If not, the development halts at the 3rd larval stage = ‘dauer’, can survive for months without food. The larva has lower metabolism, limited protein synthesis and survives on fat stores. The larvae will quickly move through next stage if conditions improve. The worms are easily genetically manipulated by being fed bacteria expressing double stranded RNA (RNAi) to genetically modify them. 300+ genes seem to be involved in lifespan regulation. It has been confirmed that major pathways are involved in the regulation of lifespan e.g. Insulin-like signalling and germline signalling. It is not known whether the worm can sense its environment by neuroendocrine signalling. Insulin-like signalling There are 37 insulin signalling family members in C. elegans. Daf-2, dauer formation gene is important in insulin signalling. Daf-2 encodes a receptor that is homologous to the IGF receptor in humans. Age-1 is an important gene that works with Daf-2 to extend life. Age-1 encoded PIK3, phosphoinositide 3-kinase involved in the insulin signalling pathway. When there is enough food, Daf-2 activation leads to PI-3K (AGE-1) activation which generates PIK3. This activates downstream kinases e.g. protein kinase B. Daf-16 (known as the FOXO transcription factor in mammals) is phosphorylated and so it cannot enter the nucleus and remain in the cytosol. Therefore it cannot inhibit the expression of reproduction and growth genes. The DAF-2 pathway, which is thought to shorten lifespan by inhibiting daf-16 activity, inhibits DAF-16 nuclear accumulation. In low food conditions Daf-2 not activated and so Daf-16 enters nucleus and represses growth and reproduction genes (and stress resistors). Stress resistance is upregulated.
Germline signalling If the germline primordial cells (PGCs) are removed by laser ablation or mutation, the worms live 60% longer. However when the whole gonad is removed the worms’ lifespan does not increase. Killing the germline precursors of daf-2 mutants causes the animals to live approximately four times as long as normal. This suggests that the germline and Daf-2 work cooperatively to regulate lifespan. A strong insulin-like signal can make up for the loss of signals from the gonads and shorten lifespan.
Drosophila Drosophila is not widely used but can be useful because they show functional senescence e.g. hearts, learn less well, explore less. They have: gene families, kidney-like structures, neurons that develop protein aggregates. Dietary restriction and low fecundity extend fly lifespan. Reducing insulin or IGF signalling in flies can increase their lifespan. Stress resistant mutants e.g. oxidative stress increase live longer
Model limitations Werner and AT mouse models Wrn-null and ATM-null mouse models do not show the degenerative pathologies. However, if the mice are crossed with telomerase-deficient mice, offspring do show degenerative ageing pathologies. Double mutants are needed in mice to display pathologies.
Carlessi L et al., 2013 present iPCs as an alternative to models. Neurospheres made from mutant cells can be used to study the disease.
WHY DO WE AGE?
Defining Ageing There are many changes that occur as an organism ages: accumulation of nuclear and mitochondrial DNA damage due to oxidative stress, gradual immunodeficiency leading to decreased immunity to infections, organs lose their ability to maintain homeostasis because signalling pathways are damaged and stochastic accumulation of tissue damage.
PROGEROID SYNDROMES Progeroid syndromes (PS) are a group of rare genetic disorders that mimic physiological ageing, making affected individuals appear to be older than they are – accelerated ageing. These syndromes can affect many organs – segmental or one organ – unimodal. Segmental progeroid syndromes: > Werner syndrome > Ataxia telangiectasia (AT) > Dyskeratosis congenital (DC) > Hutchinson-Gilford progeria syndrome (HGPS) Unimodal progeroid syndromes: > Familial Alzheimer’s disease > Familial Parkinson’s disease
I. Werner syndrome WS is caused by an autosomal recessive mutation and affects 1/100,000 people. Sufferers develop normally until puberty when they stop growing and show premature ageing. Symptoms: cataracts, grey hair, osteoporosis, cancer, atherosclerosis, poor glucose regulation, immunodeficiency, osteoporosis, skin atrophy, myocardial infarction, brain symptoms are rare. The median age of death is 47-48 years old caused by myocardial infarction and cancer. Mutations in the WRN gene lead to proteins without a nuclear localisation signal (NLS). The protein is a RecQ helicase enzyme that unwinds DNA and is involved in genome maintenance and stability. Wrn proteins are involved in DNA repair, recombination and telomere maintenance. The helicase unwinds the DNA in a 3’-5’ exonuclease activity. Werner cells in vitro undergo telomere shortening, fewer divisions, premature senescence and incorrect recombination events – translocations and deletions. Cells treated with H2O2 will initiate DNA damage signals and trigger cell senescence as Werner cells. Werner cells have different responses to oxidative stress.
II. Ataxia telangiectasia (AT) AT is caused by a rare autosomal recessive mutation in the ATM gene. The ATM protein is a kinase that phosphorylates proteins involved in DNA damage signalling, DNA repair and telomere maintenance. AT cells show telomere shortening, genome instability and premature senescence. It is a progressive cerebellar degeneration – the Purkinje cells die leading to ataxia. Telangiectasia – tiny red veins develops and the skin develops pigment abnormalities and hair greying. Melanocytes cannot cope with accumulating DNA damage and so die, and so not enough melanin is produced. Other symptoms include immunodeficiency and a wide range of aggressive malignant tumours such as leukaemia and lymph gland cancer. Ataxia is the characteristic symptom – uncontrolled muscle movement. III. Dyskeratosis congenital (DC) DC is a rare, inherited, usually male disease that causes the degeneration of mucous membranes, teeth, nails and skin pigmentation. Patients can develop cancers, grey hair, and osteoporosis and have bone marrow dysfunction. 6 DC genes have been identified that are all involved in telomere maintenance (telomerase components or telomere cap). The genome becomes unstable when these genes are mutated. Agarwal et al, 2010 in Nature Fibroblasts from DC patients were transduced with a cocktail of transcription factors (Oct5, Klf4, Sox2 and c-Myc) to induce them into a pluripotent state. The iPS cells were found to have longer telomeres showing that the upregulation of the telomerase RNA component (TERC) is part of pluripotent state. When these cells differentiate, TERC levels fall and telomeres become shorter. Several telomerase components are targeted by pluripotency-associated transcription factors.
IV. Hutchinson-Gilford progeria syndrome (HGPS) HGPS is a caused by a dominant deletion of 50 amino acids in the lamin A, a protein that forms part of the lamin mesh underneath the nuclear envelope. The protein is truncated and so it is incorrectly spliced and processed – it stays farnesylated. Farnesyl is a 15 carbon lipid molecule. This mutant ‘progerin’ protein accumulates in cells. It affects the nuclear envelope and chromatin because lamins have both structural and signalling roles involving the Wnt pathway. The incorrect interaction between the progerin and genome leads to DNA damage and senescence. Does the lamin directly interact with chromatin and influence gene expression e.g. of Notch effectors? HGPS cells in culture have a reduced lifespan and display irregular nuclear phenotype: chromatin organisation is altered there is less heterochromatin, blebbing and binucleated cells. Telomere lengths are reduced and telomeres aggregate. The response to the DNA damage is chronic as the progerin lamin mesh is not changed. Progerin signals to p53 and Rb pathways that lead to cell cycle arrest and early cell senescence. Non-HGPS patients will produce more progerin as they age, Sufferers develop symptoms before the age of two. Sufferers have very thin skin; they lose subcutaneous fat, alopecia, stiff joints, and osteoporosis and heart degeneration. The mean age of survival is 13 years usually caused by a heart attack or stroke.
Hernandez et al., Dev. Cell, 2010 The defects are associated with inhibition of canonical Wnt signalling, due to reduced nuclear localisation and transcriptional activity of Lef1, but not Tcf4, in both mouse and human progeric cells. LEF-1/TCF transcription factors mediate a nuclear response to Wnt signals by interacting with beta-catenin. Defective Wnt signalling, affecting ECM synthesis may be critical to the aetiology of HGPS because mice exhibit skeletal defects and apoptosis in major blood vessels proximal to the heart. This might explain why progeria patients have skeletal problems and thinning/apoptotic blood vessels. This could also be the reason why old blood vessels can thin. HSPG models The knockdown of Zebrafish lamin A/C induces laminopathies associated with muscular dystrophy and craniofacial abnormalities. 2013 clinical trial Lonafarnib is a small drug that has been trialled to help patients with HGPS. It is an inhibitor of farnesyltransferase (FTase). This enzyme adds the farnesyl lipid moiety to lamin A proteins. 25 children with HGPS participated in the open-label trial. There was body weight gain, the arteries appeared to be less stiff (as judged via measurements of carotid-femoral pulse-wave velocity), and bone rigidity measurements and bone mineral density were improved relative to before drug treatment. DNA damage Progeroid syndromes have one thing in common – they lead to genomic instability. They suggest that genomic instability is a key factor in ageing. Instability e.g. DNA damage, telomere integrity, etc. is dangerous because it leads to cancer, senescence and cell death. Stress responses will be initiated to try and repair the damage. Cells can only cope with a certain amount of DNA damage and will eventually apoptose or enter quiescent. However do progeria syndromes really mimic aspects of ageing? Evolutionary aspect It may be that there are mechanisms that have evolved over time to ensure survival and maintenance of reproductive organs until reproductive age. Past this age, there is no selective advantage to keep these mechanisms and this may explain ageing and the degeneration of tissues and organs. However there may be selective advantage for older people to look after their grandchildren freeing up energy and time to produce more offspring. STUDYING AGEING The ageing process is gradual and heterogeneous. The ageing rate is affected by both environmental and genetic factors. There are no biomarkers that can be used to study ageing and the changes in brings. Model organisms are used but there are advantages and disadvantages to using them. Ageing is very expensive and difficult to study, especially in humans. Subjects will need to be followed throughout their lifetime. This is expensive even in animal models which live for a long time too: Yeast (Saccharomyces cerevisiae) – 3 days, 25-35 generations Caenorhabditis elegans – 12-20 days Flies (Drosophila) – 50-80 days Rodents – less used, 18 months-4 years Humans – up to 80-100 years
Therefore there are very few cohort studies. Cohort studies are difficult to standardise because there are so many variables to control e.g. different genes and environment of each participant and different ageing rates of different organs. Also different participants will have different ageing dynamics, all these variables lead to noise. These model organisms are cheaper and have a shorter timescale. Furthermore the variables are easier to control in a laboratory environment. Observed changes in physiology may not be harmful and preventing them in experiments may be detrimental to the organisms’ health.
Ageing involved the gradual accumulation of changes on different levels at different rates because the rate of ageing is not always the same in any individual. Each tissue will respond to ageing in its own way: muscle and fat tissues undergo atrophy; wound healing and the immune response become compromised. Ageing is a major risk factor for many different chronic and fatal diseases e.g. cardiovascular diseases, arthritis, Type II diabetes, cancers… Ageing is very economically costly as health, transport and social infrastructure has to be specially modified to cope with the unique demands old age brings.
MODEL ORGANISMS Budding yeast Yeast is a simple organism and has been a staple model organism. Its small genome has already been sequenced. They are quick and cheap to culture, easily genetically manipulated e.g. knockout strains, and analysed. There are many gene orthologues in humans, so any findings in yeast may be translatable to humans. Yeast have a finite replicative capacity = ‘replicative lifespan’ (RLS) and they also have a limit to how long they can survive in a non-dividing state = ‘chronological lifespan’ (CLS). The molecular basis of RLS and CLS can be studied. Different yeast strains can be cultured in defined conditions. Old yeast display many physiological changes. They are much larger and their cell cycle and protein synthesis is slower when compared to younger yeast cells. The cell surface appears ‘loose and wrinkled’. Old yeast also have bud scars. Old cells express some apoptotic markers. Yeast are an important model organism because their ageing biology has been conserved in humans, showing that there are fundamental features of ageing present in most organisms. Important biomolecules SIRTUINS – silent information regulator 2 family of protein deacetylases/Sir2 proteins Sir2 proteins are an ancient and highly conserved family of proteins found in all forms of life and display a structure and catalytic function that have been maintained from bacteria to humans. Sir2 proteins are NAD-dependent deacetylases that were originally discovered to regulate longevity in budding yeast by Kaeberlein et al, 1999 and Imai et al, 2000. NAD is an electron carrier in the electron transfer chain. Sir2 proteins are involved in gene silencing by chromatin deacetylation. They also act as nutrient sensors and regulate many aspects of physiology. Sir2 proteins set the rates of cell division, metabolism and protein synthesis. Deletions of Sir2 have shown to shorten the replicative lifespan of yeast. Overexpression increases the RLS. Nutrient sensing and ageing (Guarente, 2013 Review) Caloric restriction without malnutrition has been proved to increase lifespan in mammals and yeast e.g. reduced glucose and/or amino acids broth for the yeast. During times of plenty, cells will have high metabolism and produce less NAD+ (more NADH and H+) as they use up NADH to produce ATP for biosynthesis and growth. Sir2 is inactive as more genes are highly active (increased protein synthesis) and the reproductive activity is high therefore the RLS is normal. Conversely during periods of low calorie input, metabolism slows down and there is more NAD+ in cells. Sir2 is active because it silences genes reduce metabolism and reproduction activity. The RLS is longer than normal.
These observations present an interesting dichotomy. Cells have a ‘choice’: to reproduce or live longer. These effects have been demonstrated in mammals, rodents, C. elegans, Drosophila and yeast. It is important to note that there have been independent groups both showing that Sir2 proteins do and do not extend life. This may be caused by differing genetic background. However the consensus is that sirtuins play a role in extending life. Sirtuins systematically redirect mammalian physiology in response to diet. Sir2 proteins reduce metabolic activity and maintain it at the baseline level and increase glycolysis efficiency.
TOR KINASES – Dibble & Manning, 2013, Nature Review Target of rapamycin (TOR) is a serine/threonine kinase that is a master regulator of metabolism and growth. It is a convergence point of a vast signalling network to sense changes in extracellular and intracellular nutrients, and responds accordingly by regulating metabolism, growth and proliferation. TOR proteins are nutrient responsive and so have increased activity when nutrients are high. A decrease in TOR activity increases RLS and CLS. Many physiological signals affect the activation status of TOR. TOR proteins receive various signals and integrate them into output signals that lead to appropriate downstream effects. TOR is involved in various signalling pathways that may be involved in the ageing process. >> When TOR activity is reduced due to nutrient starvation, stress response factors enter nucleus, leading to resistance of oxidative and temperature stress. Experimentally, the addition of rapamycin will lead to these downstream effects. >> In mitochondrion–to-nucleus signalling, in response to mitochondria dysfunction TOR can decrease RLS in yeast. >> TOR represses autophagy and this can reduce yeast CLS. Autophagy is a starvation response, where cells digest non-essential organelles as an energy source. >> TOR is involved in coordinating metabolic changes in response to nutrient changes e.g. in yeast: a change to respiration from fermentation when cellular glucose levels falls. This may increase RLS and/or CLS. >> TOR promotes transcription of ribosomal proteins (RPs) and rRNA processing factors. If a cell is starving or TOR is inhibited transcription is greatly reduced and translation is impaired. This leads to decreased ribosome biogenesis and protein translation. Conclusions from yeast studies Yeast replicative lifespan and/or chronologic lifespan can be extended via Sir2 overexpression, TOR-inhibition, Ch9/Akt (later) or dietary restriction. The genetic mechanisms behind these processes are conserved in worms, and flies and mammals. However there are limitations to using yeast as model organisms for humans. It is not known just how much of yeast biology is conserved and therefore applicable to humans. Yeast have different DNA modifications e.g. no methylation, no telomere shortening with age.
C. elegans as a model organism Much is known about the nematode worm. The 1mm long adult has exactly 959 cells whose exact lineage from zygote to somatic cells has been mapped. The nematodes are very short-lived and easily cultured, and the genome has been sequenced too and there are many genetic resources available. Therefore data is obtained very quickly. The nematode shares orthologues with other species but lacks some crucial families e.g. Shh. Nonetheless it undergoes functional and ageing senescence as mammals and humans do. Old worms have muscle atrophy (sarcopenia), reduced skin elasticity and vulnerability to infections. If there is enough food, the development of egg to adult occurs in 3-4 days. If not, the development halts at the 3rd larval stage = ‘dauer’, can survive for months without food. The larva has lower metabolism, limited protein synthesis and survives on fat stores. The larvae will quickly move through next stage if conditions improve. The worms are easily genetically manipulated by being fed bacteria expressing double stranded RNA (RNAi) to genetically modify them. 300+ genes seem to be involved in lifespan regulation. It has been confirmed that major pathways are involved in the regulation of lifespan e.g. Insulin-like signalling and germline signalling. It is not known whether the worm can sense its environment by neuroendocrine signalling. Insulin-like signalling There are 37 insulin signalling family members in C. elegans. Daf-2, dauer formation gene is important in insulin signalling. Daf-2 encodes a receptor that is homologous to the IGF receptor in humans. Age-1 is an important gene that works with Daf-2 to extend life. Age-1 encoded PIK3, phosphoinositide 3-kinase involved in the insulin signalling pathway. When there is enough food, Daf-2 activation leads to PI-3K (AGE-1) activation which generates PIK3. This activates downstream kinases e.g. protein kinase B. Daf-16 (known as the FOXO transcription factor in mammals) is phosphorylated and so it cannot enter the nucleus and remain in the cytosol. Therefore it cannot inhibit the expression of reproduction and growth genes. The DAF-2 pathway, which is thought to shorten lifespan by inhibiting daf-16 activity, inhibits DAF-16 nuclear accumulation. In low food conditions Daf-2 not activated and so Daf-16 enters nucleus and represses growth and reproduction genes (and stress resistors). Stress resistance is upregulated.
Germline signalling If the germline primordial cells (PGCs) are removed by laser ablation or mutation, the worms live 60% longer. However when the whole gonad is removed the worms’ lifespan does not increase. Killing the germline precursors of daf-2 mutants causes the animals to live approximately four times as long as normal. This suggests that the germline and Daf-2 work cooperatively to regulate lifespan. A strong insulin-like signal can make up for the loss of signals from the gonads and shorten lifespan.
Drosophila Drosophila is not widely used but can be useful because they show functional senescence e.g. hearts, learn less well, explore less. They have: gene families, kidney-like structures, neurons that develop protein aggregates. Dietary restriction and low fecundity extend fly lifespan. Reducing insulin or IGF signalling in flies can increase their lifespan. Stress resistant mutants e.g. oxidative stress increase live longer
Model limitations Werner and AT mouse models Wrn-null and ATM-null mouse models do not show the degenerative pathologies. However, if the mice are crossed with telomerase-deficient mice, offspring do show degenerative ageing pathologies. Double mutants are needed in mice to display pathologies.
Carlessi L et al., 2013 present iPCs as an alternative to models. Neurospheres made from mutant cells can be used to study the disease.
WHY DO WE AGE?
