AGEING

AGEING

1. DEFINITION

   • Aged tissues and cells have less resilience to stress
     • Lose the ability to maintain homeostasis
     • Lose ability to repair damage
       • Ageing is a major risk factor for chronic/fatal diseases e.g. Cardiovascular disease, arthritis, T2 diabetes, pulmonary disease and cancer
       • Non-aged cells have mechanisms of genome maintenance that quickly repair damage & have checkpoints to monitor genome stability
         • DNA damage repaired via stress response
           • Stops cell cycle and triggers repair
             • If repair is not possible, cell is tipped into senescence or apoptosis
   • "Gradual changes in structure of any organism that occurs with the passage of time that do not result from disease or other gross accidents that eventually lead to the increased probability of death as the individual grows older"
     • Miller-Keene Encyclopedia and Dictionary of Medicine, Nursing Allied Health (2003)
   • "The process of growing old"
     • Oxford English Dictionary
   • "A persistent decline in the age-specific fitness components of an organism due to internal physiological degeneration"
     • Rose (1991)

2. SKELETAL MUSCLE

   • Over 50 years, muscle strength and capability begins to be lost
     • Organisation of muscle cells and connective tissues declines as variability in fibre size increases
       • Fibres become more disorganised
       • Large extracellular spaces form
       • Proteins aggregate within interstitial matrix
       • Increased infiltration of non-contractive material e.g. fat & connective tissue
         • Muscle-to-fat ratio changes, leading to metabolic syndrome & insulin resistance
           • removes key protein synthesis signals
             • linked to Type 2 diabetes
               • PANCREAS INFO
           • Fat can trigger inflammation, generates ROS and releases free fatty acids into the blood
     • Mitochondria undergo changes due to ageing
       • Fewer & larger
         • Shown by EM
         • Vacuolisation of matrix occurs & cristae shorten
       • Muscle ATP production declines by 5% per decade
         • less mtDNA/ increased damage to mtDNA not so readily repaired?
         • More protein oxidation & lipid peroxidation?
         • Produces enough energy for basal activity but not for stress/exercise
         • Declining circulatory system can result in less nutrient delivery
     • Drugs taken for other conditions may have disadvantageous effects on muscle
       • e.g. steroid medication - Prednisone (treats certain types of arthritis) e.g. statins (lower cholesterol levels)
     • Muscle satellite cells
       • Adult myofibrils are terminally differentiated, post-mitotic cells
         • Pax7+ satellite cells can be activated & driven out of G0 to divide & fuse with damaged muscle fibres to regenerate myonuclei
           • Aged rodents and humans have been found to have fewer satellite cells that can divide less than in young tissues
         • More fibrogenic than myogenic
           • In vitro, old rodent satellite cells produce less organised and more fragile myotubes that are more fibrogenic than myogenic and apoptosis more often
           • Decreasing canonical Wnt signalling prevents switch to fibrogenic lineages
             • Brack et al (2007) showed that a decreasing canonical Wnt signalling can prevent aged satellite cells switching from myogenic to fibrogenic lineages as they proliferate.  C1q complement in old serum also activated canonical Wit pathway, suggesting that this external signal is involved in ageing the niche
       • Cell niche undergoes changes as the organism ages
         • SC NICHES
         • Young
           • SC in close contact with immediate components of niche that influence its behaviour
             • Basal lamina
               • Locational info & functions as reservoir for several growth factors
             • Fibroblasts & myofibrils secrete paracrine factors
             • Blood vessels & nerves also regulate SC activity
         • Aged
           • Changes to myofibre alter signals sent to SC
           • Basal lamina thickens due to fibrosis
             • Affects SC function
           • Systemic environment in niche shifts towards inhibitory influence on SC
             • Niche also disrupts SC quiescence
               • Aged muscle fibres express more FGF2
                 • Chakkalakal et al (2012) showed that aged muscle fibres express more FGF2, which drives satellite cells out of quiescence and depleted them Perhaps the muscle fibre is expressing more FGF2 to try to change age-related changes?
       • Young blood found to rejuvenate aged satellite cells
         • Manipulate therapeutically to maintain strength in old muscles?
   • SARCOPENIA
     • Loss of muscle mass/cells and change in strength and function that causes frailty
       • Frailty = exhaustion, reduced physical activity, slow walking, reduced grip strength. 
       • Causes
         • Endocrine
           • e.g. loss of anabolic testosterone 
         • Nutrition
         • Inactivity
           • exercise can help e.g. increase ROS scavengers
           • Weight bearing exercise are v effective at decreasing frailty
         • Inflamatory process
           • e.g. IL-6 increases
           • Therapies could be tried that reduce inflammation against chronic infections e.g. CMV, hormonal & nutritional interventions
         • Increased protein turnover
         • Reduced activity of satellite cells
           • Fuse with muscles and generate new myofibres

3. CV SYSTEM

   • Aged hearts have reduced function and the body stresses the system
     • Reduced IGF-1 signalling improves cardiac performance in old Drosophila and reduces age-associated cardiomyocyte dysfunction in mice BUT low serum IGF-1 levels increased risk of heart failure in elderly humans.  GH can also improve heart failure recovery and reduce age-associated diastolic dysfunction
     • Tissue is less organised
     • Fibrous ECM laid down
       • L ventricle walls thicken causing ventricle filling to decline
         • Exercise intolerance results - person unable to exercise due to pain, dizziness, breathlessness etc
     • Telomeres shorten
     • Strength of contraction decreases
     • Age-associated cardiomyopathy
       • Studies in old mouse hearts have found fibrosis, vacuolisation of cytoplasm, variable and hypertrophic myocyte fibre size, collapse of sarcomeres, mineralisation and arteriolosclerosis
   • Mitochondria
     • Increased protein oxidation
     • more mtDNA damage
     • Increased mt biogenesis
     • More ROS released
       • ROS scavenging systems e.g. SOD convert superoxide to safer H2O2
         • SOD = superoxide dismutase deactivated ROS by catalase or glutathione peroxidase or peroxiredoxins
         • SOD1 found in mt matrix and SOD2 in cytosol
       • ROS causes mtDNA lesions
         • mtDNA thought to be near the IMM ROS creates lots of mtDNA lesions e.g. oxidised DNA bases, basic sites and single/double stranded breaks
         • No protective histones in mtDNA, so is vulnerable
         • Repair inc. direct reversal, base excision repair, mismatch repair and ds break repair
     • decrease in mt SERC2 protein, regulates Ca mediated coupling of heart cells
       • SERC2 = sarcoplasmic reticulum adenosine triphosphatase
     • Increased mt permeability transition pore in IMM
       • Leads to mt swelling, collapse of membrane potential and ATP depletion
         • Apoptosis triggered
   • Aged heart SCs
     • Presence is controversial
     • More likely to go into programmed cell death and less resilient in older mice/humans than younger
     • More apoptosis, shorter telomeres and increased senescence
       • 14C labelling in human hearts showed that the turnover of renewal rate of cardiomyocytes at 25yr was 1% and decreased to 0.45% at 75yrs
   • Arteries/capillaries also affected
     • Will be stiffer due to cross-linked ECM and dilated. Also affects mechanosensitive gene expression Endothelial progenitor cells less active when old, perhaps due to inflammation and down regulation of IGF/insulin signalling?
   • Vinculin upregulated in aged hearts allows retention of efficient contraction and power for longer
     • Vinculin is part of the adhesion complex that interacts with cytoskeleton Study looked at rodents, monkeys and drosophila and found conserved results. 

4. CALORY REDUCTION

   • Improves CV health by reducing cellular activity
     • Less damage to organs due to ageing
       • Directly by up regulating eNOS & reducing endothelial ROS & pro-inflammatory pathways
       • Indirectly by reducing serum cholesterol, triglycerides, fasting glucose and fasting insulin levels
         • Lower risk of arteriosclerosis and reduces blood pressure

5. BRAIN

   • Cognitive decline with age
     • 50% of >85 have Alzheimer's
     • Aged brain has less coordinated activity across brain areas
       • Aged people with best cognitive abilities can recruit other brain areas during tasks even if brain is damaged e.g. AD
       • Difficult to tell difference between normal aged brain and pathological aged brain
     • Due to changes in
       • synapses
         • Balance of inhibition and excitation could be altered, excitotoxicity and neurodegeneration could occur
       • myelination
         • could be involved in coordination of areas
       • Epigenetics
         • oxidative stress could change epigenome and silence genes. In mice a HDAC inhibitor can mimic environmental enrichment, promoting neuronal plasticity and recover memories
       • Autophagy
         • decline, protein aggregates increase
         • Damaged mt must be cleared away as can release ROS or pro-apoptotic signals e.g. cyt-c
         • Several mechanisms
           • Molecular chaperones & proteases
             • Fuse with mt causing damage/fragmentation, can be degraded through autophagosome formation and lysosomal degradation
           • LAMP-2a can decline with age
             • Important factor in autophagy
         • Clearing senescent cells can reduce ageing
           • Senescent cells secrete growth factors, cytokines and proteases, not passive Buildup = bad as contribute to inflammation and other signalling processes DNA damage activates GATA4, activates NF-kB to trigger inflammation and senescence GATA4 accumulates in ageing tissues, including the brain 
           • Progeroid mouse models, senescent cells labelled & killed
             • Prolonged health of fat, skeletal muscle and eyes
           • GATA4 accumulates in ageing tissues e.g. brain
         • mTOR signalling inhibits autophagy in nutrient-rich conditions
           • Evidence linking autophagy & ageing
             • C. elegant long-lived mutants have mutations in autophagy-related genes
             • Drosophila mutations in Atg8a reduces lifespan and over expression in nervous system extends lifespan by 56%
               • Atg8a = autophagy gene
             • KO mice for some autophagy genes show neurodegeneration
         • drugs
           • rapamycin inhibits TOR,stimulates autophagy and extended median lifespan in drosophila by 22%
             • useful in neurons as autophagy is highest near the nucleus where aggregate to normal protein ratio is the highest (rapamycin has side effects including immunosuppression)
           • LICL activates autophagy by inhibiting IMPase
             • mTOR-independent IMPase = inositol monophosphatase  reduces inositol levels 
             • reduced neurodegeneration in drosophila AD models
             • delayed progression in ALS mouse models &patients
   • Reducing mitochondrial activity could lower neuronal stress
     • Mt is where nutrients are oxidised. electrons from NADH and FADH2 flow down ETC which si coupled to electrochemical gradient generation across the inner membrane (complexes I, III & IV) complex V uses this to generate ATP
     • Reduced expression of ETC in adult neurones extended lifespan in Drosophila
       • Decline in mt activity in human brain an attempt to protect it from stress?
     • Risk factors for early onset Parkinsons are mitochondrial
       • e.g. Mutations in
         • DJ-1 redox sensor (antioxidant protein) that quenches ROS
         • PINK regulates mt mediated apoptosis
           • PINK = PTEN-induced putative kinase 1
         • Parkin (E3 ubiquitin ligase) that maintains mt function and reduces oxidative stress
           • sits in outer mt membrane

6. CAUSES

   • Clues from progeroid syndromes
     • Segmental
       • Werner Syndrome
         • Autosomal recessive syndrome caused by mutation in WRN
           • Usually the mutation is in the nuclear localisation signal
           • WRN is a RecQ DNA helices
           • Usually maintains genome stability as is involved in repair of oxidative damage
             • Prevents polymerase skipping '8-oxo-dG' damage
             • Cultured cells are fine until stressed with H2O2
         • Characterised by normal development until puberty, then
           • Cataracts
           • Grey hair
           • Osteoporosis
           • Atherosclerosis
           • Cancer
           • Poor glucose reguation
           • Immune changes
           • Skin atrophy
           • Myocardial infarction
             • Patients die at 47-48 yrs due to myocardial infarction or cancer
         • Heterochromatin alterations are a key driver of human ageing
           • Zhang et al. generated WRN KO human ES cell lines and differentiated them to mesenchymal SCs to look at the chromatin and saw the organisation and interactions with the lamina were altered WRN KO MSCs behaved like aged cells in vitro as they proliferated less, had shorter telomeres and unregulated ageing and senescence markers and in vivo they could produce fewer muscle cells than WT MSCs
       • Ataxia telangiectasia
         • Characterised by progressive cerebellar degeneration (Purkinje cells, ataxia), skin abnormalities e.g. telangiectasia Itiny red veins, atrophy, pigmentary abnormalities etc) and malignant tumours like leukaemia Purkinje have 2/3 copies of genome, are branched so may be more vulnerable to AT
         • Rare autosomal recessive syndrome caused by mutations in ATM gene
           • Codes for protein that phosphorylates proteins involved in DNA damage signalling, repair and telomere maintenance
             • AT cells have shorter telomeres, genome instability and premature senescence iPSCs programmed to have AT and showed X-ray sensitivity, chromosomal aneuploidy and cell death
           • Breakdown of DNA damage repair is involved in ageing
       • Dyskeratosis congenital
         • Mucous membranes, teeth, nails and skin pigmentation affected Patients can have cancer, grey hair and osteoporosis, although most die of BM failure as HSCs are bad at self-renewal. 
         • 9+ genes associated with DC, involved in telomere homeostasis
           • Telomeres are TTAGGG repeats that expand over >10kb at the end of each chromosome and function to protect chromosomal ends to prevent instability. Everytime a cell divides a mechanism is needed to stop telomeres shortening
         • Problems in telomere homeostasis involved in ageing
       • Hutchinson-Gilford progeria syndrome
         • Usually develops in first 2 years of life and mean survival is 13 years, when most patients die of heart attack or stroke
         • Caused by dominant mutation in laminin A
           • 50aa deletion and is incorrectly spliced, leading to accumulation of protein progerin
             • Nuclear membrane collapses inwards slightly, affecting many pathways
               • cells have reduced lifespan, blebbing, altered chromatin organisation and binuclear cells. Also reduced telomere lengths, elevated DNA damage and progerin signalling to p53 and Rb pathways causes early cell senescence. Also leads to defective ECM
               • e.g. entry of beta catenin to nucleus
         • Lonafarnib (small molecule drug) inhibits FTase
           • FTase = farnesyltransferase
           • Trial in 25 HGPS children
             • Body weight gain, less stiff arteries, improved bone rigidity and mineral density
               • Strokes and headaches appeared to be reduced also
         • Progerin protein involved in ageing
       • Bloom syndrome
       • Cockayne Syndrome
         • Progressive neurological degeneration healing loss and cataracts Mean lifespan is 12 years CS patients are hypersensitive to UV
         • Caused by faulty DNA repair
           • Mutation in CBA or CSB gene
             • CB = cockayne syndrome complementation group genes 
             • DNA wraps around CSB homodimers and CSA is part of ubiquitin ligase complex
             • Oxidative damage to DNA bases in strands that ar being transcribed can't be repaired
               • Blocked transcription can trigger apoptosis
                 • High fat diet found to reduce this in mice
     • Unimodal
       • Familial Alzheimer's disease
       • Familial Parkinson's disease
     • SYNDROME X
       • Brooke Greenberg did not age past 11 months but lived to be 20 years old
       • Telomeres matched age and no mutations in progerin or WRN
   • Evidence suggests DNA DAMAGE THEORY OF AGEING
     • Loss of genome stability is key to ageing
     • HDAC
       • Decreased HDAC1 function increases sd breaks
         • increases neuronal death and accelerated neurodegeneration as DNA is more open to damage
           • Manipulating HDAC1 activity could be therapeutic?
       • SIRT1&SIRT6 required for efficient ds breaks
         • NAD+ dependent HDACs
         • SIRT6 KO mice had defective repair of oxidatively damaged bases
           • Died soon after weaning with several progeroid syndromes inc curved spine, decreased bone density, lymphopenia and severe metabolic defects
     • ROS
       • ROS = molecules with an unpaired electron in the outer orbit and are caused by e.g. UV light ionising radiation, chemicals, toxins, leakage from ETC in mt, cut p450 metabolism, peroxisomes and synthesis by phagocytes. 
       • Damages DNA by forming 8-oxo-G
         • Also oxidise polyunsaturated fatty acids to make them a radical which can covalently bind with other fatty acid side chains and change the lipid shape & properties. 
         • Pairs with C and A, causing GC to AT transversions
     • Intrinsic/extrinsic sources lead to DNA damage
       • Repair pathways interrupted/blocked
         • Mutations, cell cycle arrest, transcription impairment, gene expression dysregulation & apoptosis
           • Cell loss/dysfunction means SCs are depleted, tissue homeostasis is disrupted
             • Ageing of organism
       • DNA repair mechanisms down-regulated in G0 SCs
         • This is as they are mostly cell cycle linked If a low fidelity repair system is used to repair then a mutation can be perpetuated and spread through a tissue
         • Telomere maintenance is v important in adult SCs
           • Carried out by telomerase (ribonucleoprotein complex)
             • TERC has RNA template for species specific telomeres
               • In humans this is around 450 nt long of AAUCCC repeats
               • TERC KO mice had dysfunctional telomeres, high rates of cell turnover, poor organ maintenance and premature ageing
                 • Phenotype reversed if mouse also Exo1 KO
                   • Exo1 = DNA/telomere repair factor
                   • Cells no longer signalled DNA damage, improved organ maintenance and lifespan with no chromosomal instability
             • TERT is telomerase reverse transcriptase unit
               • Overexpressing mice also expressing tumour suppressors had 26% longer median lifespan
           • DISPOSABLE SOMA THEORY
             • No telomerase, chromosomes would shorten with each division as DNA polymerase cannot fully replicate lagging DNA strand
               • Cells would have a 'mitotic clock' of replicative senescence
                 • Short telomeres would activate DNA damage signals including p53 pathways and lead to cell cycle arrest and/or apoptosis
           • Telomeres could provide biomarkers for ageing
   • MITOCHONDRIAL FREE RADICAL THEORY OF AGEING
     • ROS generated in respiration causes macromolecular damage and ageing
       • As we age ROS damage increases, but mixed evidence as to if it actually causes ageing
     • FOR
       • C.elegans mutants
         • Long-lived C.elegans with altered complex III or no CoQ
           • isp-1 mutant (altered complex III) lk-1 mutant (lack coQ for complexes I & II)
           • RNAi showed reduced components results in longer-lived worms
             • Reduced ETC activity can extent life?
         • Shorter lifespan in mev-1 mutant (mutation in complex II)
           • More ROS
         • BUT may be due to pathology NOT premature ageing
           • C. elegans = anaerobic, can't compare to aerobic
             • SOD2 KO worms may live longer, but could be severely life-shortening in SOD2 KO flies/mice
       • Indirect alteration/reduction in ETC protected mouse from diabetes & obesity
         • e.g. tissue specific deletion of apoptosis inducing factor, stabilises ETC
           • reduced ETC function in muscle/liver
         • Reduced MCKL1 activity reduced ETC and increased lifespan
           • MCKL1 = mt ETC coenzyme for ubiquinone synthesis
         • Mice with disrupted SURF1 had increased longevity
           • SURF1 = putative complex IV assembly factor
       • Direct alterations to ETC in neurons decreased oxidative stress and amyloid plaque load in an AD model
         • e.g. COX10 deletion, complex IV subunit
       • Reducing ETC could increase mt biogenesis & turnover, alter ageing nuclear gene transcription
         • (ROS release NF-kB from inhibition and alter pro-apoptotic genes) Could explain the mixed results, as not all ETC reductions can be tolerated so some ROS is needed
     • AGAINST
       • Overexpression of SOD1, SOD2 & catalase increases invertebrate lifespan but not in mice
         • These are anti-oxidants
       • GPx4 heterozygous mice had longer lifespans but higher levels of oxidative damage
         • GPx4 is an antioxidant that reduces membrane bound lipid hydroperoxides
       • Naked mole rat
         • v high ROS levels
           • resistant to oxidative damage and no cancer
       • Increasing ROS in mice can fail to shorten lifespan
       • Ames dwarf mice have longer lifespans but higher mt ROS
       • ROS could be useful
         • Signal mt are v active
           • Message to nucleus to up transcription/translation?
   • GENETICS
     • Centenarians
       • Difficult to study as no control group
         • Compare offspring with offspring of those who had an average lifespan?
       • Ashenazi Jews >100yo
         • Sue et al (2008) age and sex matched groups and looked at offspring who were about 70 yo
         • Female offspring had 35% higher serum IGF1
         • Partial loss of function in IGF1 R (reduced phosphorylation of AKT)
       • Japanese
         • 122 105+ yo compared to 122 healthy younger controls
           • INSR type v common in the group
         • 293 95yo compared to 603 75yo
           • Pawlikowska et al (2009)
           • intronic SNPs in AKT1 & FOXO3A significantly more common in aged group
         • mean 98 yo men had 3 SNPs in FOXO3 gene that were v common
           • less cancer & CV disease, felt healthier, higher physical/cognitive function than those who died at 77yo
           • Several biomarkers for increased insulin sensitivity
     • Twin studies suggest there is up to 40% genetic component to ageing

7. CELL SIGNALLING

   • MODEL ORGANISMS
     • Yeast
       • Advantages
         • Simple, quick, cheap, easily genetically manipulated
           • small sequenced genome
         • Many gene orthologues in humans
       • Disadvantages
         • Finite replicating capacity
           • Replicative lifespan
         • limit to how long they can survive in a non-dividing state
           • chronological lifespan
         • No methylation and no telomere shortening with age
       • old yeast has slower cell cycle & protein synthesis, loose & wrinkled cell surface & some apoptotic markers
         • if apoptosis is stopped, they can resist ROS stress death
       • Mutation in glucose sensing pathway can alter CLS/RLS
         • e.g. Ras2 deletion (minics low glucose/receptor activity) increases CLS 2x
         • nutrients bind Gpr1 receptor and activate kinases
           • Phosphorylate & inactivate TFs to downregulate some gene activity & upregulate stress resistance
       • DR
         • reducing glucose/aas in broth
         • Sirtuins deacetylate histones and compact DNA
           • Sirtuins = silent information regulator 2)
           • Also deacetylate TFs, so regulate gene expression
           • nutrient sensors, regulated by NAD+
             • +++ food = high metabolism --> less NAD+
               • Sir2 inactive
                 • Lifespan/fecundity normal
             • --- food = low metabolism --> more NAD+
               • SIR2 activated
                 • Triggers stress response, reduces fecundity & increases lifespan
                   • Choice between reproducing & living longer
     • Dietary restriction can increase lifespan
       • TOR kinase (nutrient responsive)
         • high nutrients = higher activity, cell metabolism, growth & proliferation
         • Decreased TOR activity increases RLS & CLS
           • TOR influences
             • Stress response
               • reduced TOR activity --> stress factors entering nucleus
             • mt-to-nucleus signalling
               • signalling in response to mt dysfunction can decrease RLS in yeast
             • Autophagy
               • TOR represses autophagy which can reduce yeast CLS
           • Translation decreases
             • Decreased translation needed for lifespan extension?
         • Target of rapamycin
         • Involved in DR response
           • TOR inhibition mimics DR & extends lifespan
           • TOR mutants not affected by DR in yeast worms & flies
       • Reduction in one/total nutrient level
         • general calorie reduction or reduction of a food group CR in rodents can extent lifespan by up to 50%
       • 1 CR study in humans
         • Civitarese et al (2007) 6 month trial of CR -25% +/- exercise muscle punch biopsies showed upregulation of SIRT1 and mt number and reduced DNA damage markers in muscle Young, non-obese adults, only short term though
     • Drosophila
       • functional senescence e.g. in hearts, learn less well and explore less as they age
       • mutants resistant to oxidative stress live longer
       • DR, low fecundity, reduced insulin/IGF signalling extend lifespan
       • mutations in nutrient sensing extend lifespan
         • 7 ligands, 1 receptor involved in nutrient sensing Mutations in InR ---> 5% longer life, reduced cardiac ageing, storage of nutrients and upregulation of SOD Mutations in chico (an IRS) leads to 48% longer life Overexpression of dFOXO in adult fat body extends life
         • TOR KO increases lifespan
     • C. elegans
       • Advantages
         • cheap to culture/work with
         • genome sequenced, orthologues with other species
           • 300 genes identified that are involved in lifespan regualtion
       • Disadvantages
         • Lacks some families e.g. Shh
       • Old worms have sarcopenia, reduced skin elasticity & vulnerable to infections
       • insulin-like signalling & germline signalling pathways involved in regulating lifespan
         • 1 insulin receptor (daf-2) activated when +++food
           • DAF = Dauer Formation gene
           • PI-3K (AGE-1) activated, generates PIP3 & downstream kinases
             • DAF 16 (FOXO in mammals) phosphorylated, prevents localisation to nucleus
               • expression of reproductive & growth genes
           • --- food, daf-2 not activated
             • daf-16 enters nucleus
               • represses growth and reproduction genes
                 • e.g. ROS scavengers SOD2/catalase, heat shock proteins, swithing to hypermetabolic state to store excess nutrients (fat/glycogen) and drug detoxifiers
                   • Reducing pathway boosts innate immunity & reduces cell senescence
           • daf-2 mutants hav 60-100% longer lifespan
             • resist behavioural decline also 
         • CR improves lifespan, nutrient excess causes obesity & metabolic problems
           • TOR
             • RNAi of TOR can increase longevity
             • TOR is Daf-16/FOXO independent
             • DR longevity needs autophagy
               • TOR regulates autophagy, poss through TFs?
     • cellular nutrient sensing regulates ageing
     • Mice
       • Ames dwarf
         • Point mutation in Prop-1 affects pituitary gland differentiation
           • small, delayed puberty
           • long-lived (males +49%, females +68%)
           • No circulating GH, no plasma IGF-1
       • Laron dwarf
         • 50% reduction in IGF-1R ---> 33% increase in female lifespan, 16% (ns) in males
       • Snell dwarf
         • Mutation in Pit-1 (downstream of Prop-1
           • No circulating GH, no plasma IGF-1
           • Longer life, but depends on genetic background
       • Reducing GH/IGF-1 signalling extends lifespan
         • Small size beneficial?
           • Lack of GH reduces blood glucose
             • reduces insulin & increases insulin sensitivity
             • Also reduces fertility and anabolism/ROS
             • Same in humans?
               • GH & IGF1 fall in ageing
                 • muscles weaken and there are changes to body fat that are similar to GH deficiency
                 • GH treatment can increase muscle mass & reduce fat
                   • GH can rejuvenate an individual, but no lifespan increasing effect in humans according to meta analysis?
                     • Improvements to heart health & exercise ability though
   • MAMMALS
     • Insulin acts as readout of nutritional state
       • Binds IR/IGF-1R/IRB
         • Ras/IRS1-4 activated
           • Phosphorylate PI3K ---> PIP3
             • upregulation of e.g. FoxO TF
       • Mutants dont have the same results as expect from model organisms
         • IGF-IR +/- mouse females were long lived
           • Not males
         • IRec KO mice have shorter lifespans due to ketoacidosis
           • degradation of insulin signalling in humans includes type 2 diabetes and cancer
             • treatments would have to be tissue specific
               • IRS-2 KO brains had an extended lifespan
                 • Taguchi et al (2007) IRS-2 = IR/IGFR adaptor Mice were overweight and insulin reisistant
                 • Reducing insulin/IGF1 signalling good for ageing brains
               • fat specific IR KO imce lived 20% longer, were leaner and had increased inslin sensitivity
     • Mammal SCs
       • Stress ---> FOXO upregulated & localised to nucleus
         • Inhibits cell cycle, regulates metabolism, resists further stress
         • regulates NSC proliferation, NSC are depleted if FOXO is reduced
       • Growth & anti-ageing pathways compete?
         • Competition for beta catenin?
           • In development, Wnt binds & prevents b-catenin degradation
             • translocates to nucleus & drives expression of proliferation, self-renewal and lineage differentiation factors
           • involved in growth and reacts to ROS in ageing

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