1
Q
Why is it difficult to study ageing?
A
it’s difficult to study aging in people as old people usually have multiple health cconditions and therefore are a source of wide variability in research
2
Q
How to define aging?
A
- mmune system not reaccting as well to disease or an overreactive system causing chronic inlammation
- young cells are better at maintaining and repairing their genomes than older cells
- shortening of telomeres as you age in somatic cells
- oxidative stress in some tissues plays an important role in aging
- senescence ; we used to think that the cells just sit there and fdo nothing but they can release pro inflamatory cytokines and stuff which can damage cells and contribute to aging
- you tend to have more fibrous tissue in your muscles → you fall over and break stuff → you get in hospital and you decline and die 🙂
- young cells and tissues can maintain homeostasis much better → as you age the balance in your cells is disturbed and your functions decline
3
Q
What is the role of fat in ageing?
A
we used to think that fat was neutral but it turns out that fat can induce damage to other cells (especially around the vital organs)
4
Q
What are some challenges for studying ageing in humans?
A
- gradual and therefore expensive (it’s quite difficult to follow somebody from their birth to their 80s
- dynamics can change (sudden decline in health)
- people age in different ways
- external and internal influences
- lack of biomarkers
- humans are complex and long lived
- model organisms
- old mouse is not the same as old human (18 months vs 95 years) so working with model organisms may be difficult
5
Q
How do your muscles change as you age?
A
- if you’re over 50 you will start losing muscle even if you lift (but still lift!)
- increased fibre size variability
- fibres are more disorganised
- larger extracellular spaces
- protein aggregates within the interstitial matrix
- inccreased infiltration of noncontracctile material
- function declines
6
Q
How do mitochondria change as the muscles age?
A
- changes in mitochondria:
- fewer and larger
- vaccuolization of the matrix
- shortened cristae
- muscle ATP production by mitochondria declines about 5% per decade
- less mtDNA and more damage
- energy for basal activity and not stress or exercise
7
Q
How can you identify satelite cells?
A
satelite cells are Pax7+ and therefore can be easily identified
8
Q
What happens to the satellite stem cell niche as you age?
A
- old satelite cells
- rodents/humans: fewer satelite cells and they can divide less
- in vitro: old rodent satellite cells produce less organized/more fragile myotubules
- more fibrogenic than myogenic
- apoptose more often
9
Q
What factor do ageing muscle fibres express and what does it do?
A
- aged muscle fibres express more Fgf2 (fibroblast growth factor)
- takes satelite cells out of quiescence and depletes them
10
Q
What other factor falls in the muscle as you age and what’s the effect of that?
A
in the old system you will get less of the Wisp and you will get disfunctional repair of the muscle
11
Q
What is parabiosis?
A
parabiosis (you take the vascular system of the old organism and connect it to the young organism and you look at what the young blood can do to the older organism
12
Q
What happened in the rejuvenated muscle niche?
A
- rejuvenate muscle satellite niche
- decrease cannonical Wnt signalling
- can prevent aged satellite cells switching from myogenic tofibrogenic lineage as proliferate
- injecting Wnt3a into repairing muscle lef to more fibrosis
13
Q
What happens to Notch signalling as you age?
A
Notch/Delta signaling down with age (controls cell proliferation, cell differentiation and binary cell fate decisions)
14
Q
Why is studying signalling pathways in ageing difficult?
A
lots of the singalling pathways that were identified to be connected to aging are difficult to play with therapeutically because they may increase the risk of cancer as they are quite often mitogenic or intefere with the immune response of the organism
15
Q
What happens to HSCs as you age?
A
- young organisms tend to make the right proportions of the hematopoeticc cells
- as you age you tend to make more of a specific lineage → aging doesn’t affect all stem cells in the same way
16
Q
What is sarcopenia?
A
- loss of muscle mass/cells
- causes frailty : exhsaution, reduced physical activity, slow walking, reduced grip strength
17
Q
What are some causes of sarcopenia?
A
- endoccrine eg lose anabolic testosterone
- nutritional
- inactivity (exercise can help e.g. to increase ROS scavengers
- inflamatory processes eg IL-6 increases
- reduced anabolism
- reduced activity of satellite cells
- diseases can also contribute to sacropenia
18
Q
can we prevent frailty?
A
- exercise doesn’t really help
- reducing inflamation
- hormonal treatment
- nutritional intervention
19
Q
What are two main factors that affect the declining CV heart as we age?
A
- two changes as we age
- heart has reduced function
- the rest of the body is ageing and stressing the CV system
20
Q
What happens to the heart as we age?
A
- fibrosis
- some dead cells
- plaques
- decrease heart rate and arythmias
- scar tissue
21
Q
Discuss the the role of vinculin in the ageing heart
A
- proteomic analysis on the heart muscle
- the cells were upregulating vinculin (Vinculinis a cytoskeletal protein associated with cell-cell and cell-matrix junctions,) and the idea was that this would improve cell to cell adhesion and help the cytoskeleton better support the heart muscle
22
Q
Discuss the effects of young Sca1+ cells on the ageing heart
A
Long term repopulation of aged bone marrow stem cells using young Sca1 cells promotes aged heart rejuvenattion
- reconstitution of aged BM cells with young Sca1+ cells
- effective homing of functional stem cells in the aged heart
- promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells
23
Q
Discuss the impact of calorific restriction in the ageing heart
A
- indirect: reduce serum cholesterol, tryglycerides, fasting glucose and facting insulin levels = lower risk of arthosclerossi reduce blood cells
- in evolution when you have periods of stress th cells are upregulating the DNA repair systems to protect themselves so that’s what it seems to be what’s happening with calorific restrictions
24
Q
What is the difference between progeroid syndromes and unimodal syndromes?
A
- progeroid syndromes (segmental) - affect many organs
- or one (unimodal)- affect just one organ
25
Name some progeroid and unimodal syndromes
- segemental progeroid syndromes:
- Werner syndrome
- ataxia telangiectasia
- dyskeratosis cognetia
- Hutchinson-Gilford
- progeria syndrome and Bloon syndrome
- unimodal progeroid
- familial Alzheimers disease
- familial Parkinsons disease
26
What is Werner syndrome?
- autosomal recessive
- rare (one in a million)
- develop normally until puberty , stop growing and start ageing
- from 30yrs cataracts, grey hair. osteoporosis, cancer, poor glucose regulation immune changes, skin atrophy, myocardial infarction
- median death around 47/8 usually of myocardial infarction and cancer
27
What is Werner syndrome?
- autosomal recessive
- rare (one in a million)
- develop normally until puberty , stop growing and start ageing
- from 30yrs cataracts, grey hair. osteoporosis, cancer, poor glucose regulation immune changes, skin atrophy, myocardial infarction
- median death around 47/8 usually of myocardial infarction and cancer
28
What is the cause of Werner syndrime?
- the cause of Werner syndrome is a DNa helicase
- sometimes there are issues with the nuclear localization singla and the.mutations can affect whether the protein can get inside the nucleus or not
29
Describe the experiment that lead to the discovery of Wrn function in human ageing
- they had a human ES cell line that lacked WRN
- they differentiated them to mesencyhhymal styem cells and looked at chromatin
- chromatin organisation and interaction with lamina can be altered
- WRN null MSCs behaved like old cells in vitro - proliferated less, shorter telomeres, upregulated ageing and senescence markers
>90% of differently methylated regions in WRN were not affected by ageing so it’s not just accelerated normal ageing
- most were in genes associated with transcription factors
- suggests Werner syndrome due to incorrect control of gene expression
30
What is Ataxia telangiectasia?
- rare autosomal recessive
- progressive cerebellar degeneration
- skin abnormalities
- pigmentary abnormalities and hair greying
- immunodeficiency
- a wide range of malignant tumours
31
What is the cause of AT?
- mutation in ATM gene - a kinase which senses the double stranded breaks in the DNa and triggers the repair adn telomere maintnance
- the kinease is also involved in chekcpoint activity during cell cycle → malignant aggresive tumours
- when dividing ATM mostly in the nuclus and when not some in cytoplasm functions to activate and coordinate singalling involved in cell cycle checkpoint. response to oxidative stress mitochondrial funtion and apoptosis/senescence
- AT cells show telomere shortening, genome instability and premature sensescence
- iPS cells with AT show X-ray sensitivity chromosomal aneuploidy and death
32
What is Dyskeratosis Congenita?
- rare, inherited, usually male
- mucous membranes, teeth, nails, skin pigmentation affected
- can have gery hair, osteoporosis and cancer
- most die of bone marrow failure
33
What is Dyskeratosis Congenita?
- rare, inherited, usually male
- mucous membranes, teeth, nails, skin pigmentation affected
- can have gery hair, osteoporosis and cancer
- most die of bone marrow failure
34
What is the cause of DC?
- 9+ DC genes: all involved in telomere homeostasis (telomere components or telomere cap)
- reminder: Telomeres = TTAGGG repeats that can expand over 10 kb to cap ends of each chromosome
- function: protect chromosomal ends, stop chromosome instability
35
Where is the telomere maintnance especially important?
especially important in cells that often divide like some adult stem cells
36
What is telomerase and what cells have it ?
- telomerase, a ribonucleoprotein complex = TERC (RNA template) and TERT (telomerase reverse transcriptase)
- human telomerase is tightly regulated
- most somatic cells don’t have telomerase
- if no telomerase, chromosomes shorten with each division
37
What is the mitotic clock of replicative senescence?
as DNA polymerase cannot fully replicate the lagging DNA → mitotic clock theory of replicative senescence
38
What happens when telomeres are short?
short telomeres will activate DNA damage signals incl p53 pathway and lead to senscence and apoptosis
39
Why do telomeres in stem cells not shorten?
stem cells tend to have special telomere maintaining components so they can protect their genome when they sit in G0 and then their telomeres don’t shorten drastically when they divide
40
What happens in TERC null and TERC overexpressing mice?
- TERC null mice: dysfunctional telomeres, high rates cell turnover, poor organ maintnance and premature ageing
- TERT overexpressing mice also expressing tumour surpressors (to stop the TERT inducing tumours) have 26%longer median lifespan
41
What is a hutchinson-gilford progeria syndrome?
- before 2yr
- mean 13 yr survival (heart attack or stroke)
42
What is the cause of HGPS?
- dominant mutation in lamin A (make the mesh under the nucelar membrane and interact with chromatin)
- deletes 50 aas
- incorrectly spliced and processed
- progerin protein accumulates affects nuclear envelope, chromatin
43
How do HGPS cells behave inculture?
- HGPS cells in culture:
- reduced lifespan
- irregular nuclear phenotypes, altered chromatin organization, binuclear cells
- reduced telomere length
- chronic DNA-damage response:
- progerin signalling to p53 and RB pathways from telomeres causes early cell senescence
43
How do HGPS cells behave inculture?
- HGPS cells in culture:
- reduced lifespan
- irregular nuclear phenotypes, altered chromatin organization, binuclear cells
- reduced telomere length
- chronic DNA-damage response:
- progerin signalling to p53 and RB pathways from telomeres causes early cell senescence
44
Where can you get ROS from?
- unpaired electron in the outer orbit
- UV light, ionizing radiation, chemicals toxins, leakage from the electron transport chain in mitochondria
- cytochrome P450 metabolism
- peroxisomes
- synthesized by phagocytes
44
Describe the physiological significance of ROS
- noramlly some amount of ROS is generally useful for regulation of transcription factors and stuff like that but too man can:
- damage DNA
- oxidise aas and enzyme co-factors
- oxidise polyunsaturated fatty acids:
- the fatty acid is now a radical and will covalently bond with other fatty acid chains changing lipid shape and properties
45
What is Cockayne syndrome?
- rare
- progressive neurological degeneration
- hearing loss and cataracts
- mean lifespan 12 yrs
- CS patients are hypersensitive to UV
- affects some proteins needed for neuron regeneration
46
What is the cause of CS?
- most have a defect in CSB and others have defective CSA gene
- DNA wrpas round CSB homodimers CSA protein is a component of a ubiquitin ligase complex
- CS can’t repair oxidation-induced damage to DNA bases in the strands of DNA that are being transcribed
- blocked transcription can trigger apoptosis
47
How does adult stem cell DNA damage happen?
- many DNA repair mechanisms are cell-cycle linked
- G0 many repair mechanisms are down-regulated so when the cell comes out of the G0 after a long time it can have more mutations
- mutations can be perpetuated and spread throughout tissue
48
What is the mitochodnrial structure and function?
- nutrients are oxidised
- electrons from NADH and FADH2 flow down the electron transport chain
- ETC coupled to electrochemical gradient generation across inner membrane
- complex V uses this to generate ATP
49
Describe ROS scavenger systems
- superoxide converted to H2o2 by superoxide disutase (SOD)
- SOD1 in mitochondrial matrix and SOD2 in cytosol
- non-enzymatic:
- hydrophilic (ascorbate and urate)
- lipophilic (flavonooids and crotenoids)
50
Why si mtDNA prone to damage?
- mitochondrial DNa loccated quite close to the complexes → can get ROS and get damaged
- ROS create lots of mtDNA lesions
- not protected by histones
- mitochodnria can’t fix all the damage
51
What is the mitochodrial free radical theory of ageing
- ROS generated during cell respiration causes macromolecular damage and ageing
- ROS damage does increase as we age
52
What si so different about ROS in mice?
- over-expression of antioxidant enzymes increases invertebrate lifespan but not mice
- GPx4 heterozygote mice (mt antioxidant enzyme) have longer lifespan despite higher levels of oxidative damage
- naked mole rat has a high ROS but can resist ageing and cancer
- increased ROS in mice models can fail to shorten lifespan
53
What is the evidence behind the free radical theory of ageing?
- decreased components of compexes of electron transport cchain and coenzyme Q increased the lifespan of the worm
- mutated complex II increased the ROS and shortened the lifespan
54
Why should you be cautious about ROS studies in C elegans
- short-life might equal pathology and not premature ageing
- C elegans live in soil with hypoxia and anaerobic energy production
- might not be that useful for human research
55
What is the future of mitochodnrial transplants?
- now you can have egg from your mum, sperm from your dad and mitochondria from a donor to escape the mitochondrial diseases
- in the future maybe if we want to slow down ageing we can have some banks of mitochondria from healthy donors and produce humans from healthier mitochondria that would age less
56
What is autophagy?
autophagy = degradation pathways that use lysosomes
57
Describe three kind of autophagy
- authophagy:
- macro - wraps in the lipid bilayer and fuses it to the lysosome
- micro - the lysosme directly internalises something without the wrapping step
- shaperone medaited - the protein that’s missfolded has a tag recognised by the shaperone, takes it to the lysossome, upon recognition the protein is threaded through to the lysosome
58
Describe macroautophagy
- formation of double-membraned ‘autophagosomes’ that fuse with a lysosome 16-2-ATG genes involved
- **metabolism (release energy)**
- proteostasis (protein aggregate clearing
- **protection (dysfunctional organelles)**
- **regulated by environmental cues like nutrient reduction**
- in general autophagy reduces with age
- do we ccarry on proliferating during stress or calm that down and just push through
59
What is the significance of proteostasis in ageing
remove damaged organelles and proteins to keep the proteome functional
60
What evidence links autophagy and aging?
- **C. Elegans: long-lived mutants include autophagy-related genes**
- Drosophila: mutations in autophagy gene Atg8a reduce lifespan overexpression in the nervous system extends lifespan by 56%
- Mice null for some autophagy genes show neurodegenration
61
What is the significance of senescent cells in ageing
- senescent cells secrete, senescence-messaging secretome (SASP) = cytokines, chemokines, extracellular matrix proteases, growth factors, and other signals
- **Mouse: labelled and killed senescent cells, saw prolonged health of fat, skeletal muscle and eye**
62
What is the significance of yeast in studying ageing
- **simple (small sequenced genome), quick, cheap, easily genetically manipulated e.g. knockout strains**
- **many gene orthologues in humans**
- **have a finite replicative capacity = ‘replicative lifespan’ (RLS)**
- **also a limit to how long can survive in a nondividing state = ‘chronological lifespan’ (CLS)**
63
What increases yeast lifespan
dietary restriction
64
What are sirtuins?
- sirtuins - energy sensors
- regulate many aspects of physiology
- can deacetylate histones and compact DNA
- can also deacetylate transcritpion factors and so regulate gene expression
65
What happens in the abundance of food?
- lots of food
- **High metabolism, less NAD+ (more NADH and H+)**
- ***sir2* inactive, normal fecundity (ability to produce offspring) and lifespan**
66
What happens in the situation of low food
- low food
- **low metabolism, high NAD+**
- ***sir2* active, stress responses, reduced fecundity**
- longer lifespan
66
What happens in the situation of low food
- low food
- **low metabolism, high NAD+**
- ***sir2* active, stress responses, reduced fecundity**
- longer lifespan
67
What is TOR and what it its significance in ageing
- **Identified target of rapamycin, TOR (Ser/Thr Kinase)**
- **TOR is nutrient responsive (high nutrients = higher activity)**
- **regulates cell metabolism including cell growth and proliferation**
68
What happens when you decrease TORs activity
- **A decrease in TOR activity increases RLS and CLS (that cell ‘choice’)**
- TOR can regulate many cellular processes
- the cells can to some extent regulate the rate of protein synthesis
68
What happens when you decrease TORs activity
- **A decrease in TOR activity increases RLS and CLS (that cell ‘choice’)**
- TOR can regulate many cellular processes
- the cells can to some extent regulate the rate of protein synthesis
69
What is the significance of IGF-1 in ageing
Insulin/IGF-1 :the first signalling pathway shown to influence lifespan
- **Yeast, C. Elegans, Drosophila, mammals: mutations that lower glucose or Insulin/IGF signalling increase lifespan**
- **CR (not malnutrition) improves life span, nutrient excess causes obesity and metabolic problems**
70
What is the effect of the nutrient sensing pathway on ageing
- mutations in the nutrient sensing steps can alter the lifespan
- at the end of that pathway you get transcription factors which then can decide if you need to upregulate processes such as DNA repair if the cell is under stress or to keep proliferating
71
Why should you be cautious about mammalian studies of CR?
- for example you can see that only females or males will respond in a predictable way
- IGF-IR+/-mice only females are loingb lived
- IRec knockout mice have much shorter lives due to ketoacidosis
- in humans deregulation of insulin signalling - type 2 diabetes and cancer
72
Discuss the significance of DR in ageing
- **DR = reduction in one/total nutrient level (not malnutrition)**
- **general calorie restriction (CR) or reduce a food group**
- soetimes when mice are prone to oveeating theen it can alter the results
- **CR in rodents = 30–40% decrease of ‘ad libitum’ levels**
- **extends their lifespan by up to 50%**
- **CR increases lifespan in most species**
73
What is the general evidence that TOR and DR are influencing ageing
- **general evidence for TOR involvement in DR response:**
- **DR experiments most often linked to TOR signalling**
- **TOR inhibition mimics DR, and extends lifespan**
- **TOR mutants not further affected by DR (yeast, worm, flies)**
74
WHat is the link between TOR and protein translation
- we would predict that obesity could increase the translation of proteins that participate in ageing
- if we have nutrients TOR up regulates translation
- during DR TOR activity reduced and translation decreases
75
What were the results of human trail about CR and mitochondria
- six moths trial of CR
- CR muscle punch bipsies showed upregulation of SIRT1 and mitochondiral number and reduced DNA damage markers
76
Why do some people live longer than opthers?
- twin studies suggest 20-30% is genetic
- very old is more heritable
77
Why are ageing studies difficult to conduct and how can we approach it
- difficult to follow poeple for decades so research is difficult
- a good idea: use offsprring of those who live very long and compare them to those with average lifespan parents
78
What did the study of Sdhkenazi Jews tell us
- age and sex mathced groups
- offspring were about 70yr
- looked at serum
- saw female ooffspring of long-lived had 35% higher serum IGF1
- sequenced the IDF1 genes
- sequenced the IGF1 R
- saw partial loss of function in IGF1 R (reduced phosphorylation of AKT) in some of the centenarians
79
What is the significance of FOXO3 in human ageing
- males from Hawaii
- long-lived men showed 3 SNPs in FOXO3 gene
- these also had less cancer & CV disease, felt healthier, had high physical and cognitive function
- the long lived men also exhibited several biological markers of greater insulin sensitivity
80
What is the significance of ApoE in ageing
- ApoE involved in transporting lipids in bloog and binds to amyloid-B peptide
- several cohort studies highlight ApoE variants
- ApoE E4 isoform in cardiovascular disease, cognitive decline, AD, increases oligomeric soluable amyloid B peptides (longevity variants often linked to cardiovascular disease and Alzheimer’s)
81
What other genes have been highlighted in the ageing studies
also LMNA and WNR and SOD1 and SOD2
82
What genes are involved in ageing
- there are studies that suggest that ageing is a polygenic process
- they discovered 330 variants that significantly disciminated between centenarians and older adults
- genes associated with cellular differentiation, developmental processes and cellular responses to stress
83
What other aspect of centenarian biology should we coonsider
- novel bile acid biosynthetic pathways are enricged in the micorbiome of centenarians
- stool samples
- bile acids made in liver modified by gut bacteria to secondary bile acids
- centenarians had more of some acids which was proposed to kill harmful bacteria
- the role of the microbiome in ageing
