Molecular Basis of Aging: Metabolism and Genome Integrity (November 2023) PDF

Molecular basis of Aging: Metabolism and Genome Integrity 2X: Fundamental Topics in Biology Aging & Disease (lecture 2) Prof. Iain Johnstone November 2023 des by Iain Johnstone and Dr Christina Elliot Aims of this lecture Appreciate the gen...

Molecular basis of Aging: Metabolism and Genome Integrity 2X: Fundamental Topics in Biology Aging & Disease (lecture 2) Prof. Iain Johnstone November 2023 des by Iain Johnstone and Dr Christina Elliot Aims of this lecture Appreciate the genetic landscape of progeroid syndromes and its link to accelerated aging Understand how changes to insulin signalling can increase longevity (at least in Drosophila and C. elegans). Understand how telomeres are shortened by cell cycles and how they are grown. Describe what the Hayflick limit is and explain its relationship to cellular senescence. Disruption in metabolism and genome integrity are the most common features of accelerated aging Defects in a very limited set of cellular functions can cause a progeroid syndromes. Aging is developmental biology: death is the endgame! Much of what we know about the molecular mechanisms of development (and aging) comes from research using model organisms. The Fly The Mouse The Worm Drosophila Mus musculus Caenorhabditis melanogaster elegans A C. elegans mutation that doubles adult life expectancy – by perturbing insulin signalling. The opposite of a progeroid syndrome!  Insights from C. Elegans- note the Dauer larva https://www.wormatlas.org/dauer/introduction/DIntroframeset.html  Age-1: a mutated gene that promotes C.elegans longevity 1988: age-1 mutant of C. elegans described The mutant doubles post reproductive adult life expectancy from ~2 to ~4 weeks. Mutant is recessive to wild type and is a partial loss of function of function. Therefore, ONE of the normal functions of WILD TYPE age-1 is to limit adult life, reducing it by ~50% (partial loss doubles lifespan!) age-1 was found to be allelic to daf-23 (dauer formation) age-1 and daf-23 are the SAME GENE; just different alleles on the same gene daf-23 make dauer larvae all the time (even with plenty food) Well nourished age 1 mutants do not make dauer larvae but do live twice as long as wild type worms as adults. This demonstrated a link between control of dauer development AND ageing. This is a NUTRIENT SENSING defect. What is known about daf 23/age 1 and dauer larvae formation?  Dauer formation is regulated by the insulin signalling pathway Insulin promotes glucose uptake from blood and conversion to glycogen. Increased fatty acid synthesis. Increased esterification of fatty acids –adipose tissue makes fats. High calorific intake- insulin Insulin signalling in vertebrates pathway is ON Go back to systems to cells for a refresher (if Low calorific intake- insulin needed)  Insulin signalling is highly conserved Phosphoinositide The insulin pathway is 3-kinase (PI3K) conserved between humans Forkhead transcription and worms. Each named factor (FOXO) proteins in the figure are Protein kinase B orthologues (DAF-18 it (Akt) orthologous to PTEN etc.) Phosphatase & tensin homolog 1. Abundant food – insulin (PTEN) signalling activates DAF-2 In mammals, (InsR) insulin 2. Activates AGE-1 (PI3K) signalling is 3. Activates AKT (Akt) regulated by 4. Turns off DAF-16 (FoxO) two receptors, 5. Normal development (not the insulin dauer). receptor and 6. Promotes fat storage in the insulin-like intestine. growth factor 1 Calorie restriction- no insulin- DAF16 on - Dauer  Does reduced Insulin signalling cause longevity in C. elegans? The mutant alleles that cause longevity are all partial loss of- function. DAF-16 mutants Complete loss causes block age-1 longevity so constitutive dauer larva DAF-16 required formation. Thus: for age-1 longevity. 1. DAF-16 OFF- normal DAF-16 does development- short life. NOT regulate glucose uptake. 2. DAF-16 ON- starved develop AKT does. as dauer larvae. 3. DAF-16 ON a bit - normal development but long lived. Downstream targets of DAF-16: key to longevity? Mole rat has Oxidants (free elevated radicals) damage chaperones. DNA – cause Chaperones mutations promote proteostasis Summary of Insulin Signalling and Longevity 1. C. elegans calorific restriction early in life promotes dauer larva formation. 2. Partial calorific restriction promotes longevity. 3. Mutants that block insulin signalling promote dauer. 4. Mutants that partially block insulin signalling promote longevity. Drosophila- same genes as C. elegans extend lifespan. Starved flies live longer Mammals Dogs with LOW IGF-1 levels live longer than other breeds (small vs big dogs) Larger animals have higher resting metabolic rates Mouse heterozygous for IGF 1 receptor 30% life extension and increased For humans it is yet to be formally demonstrated Complete block of insulin signalling- diabetes- when untreated is fatal Very little evidence to suggest calorific restriction in humans promotes longevity Protective effects likely come from calorie/ weight management. Obesity highly correlates with T2D, increased cancer risk etc. It is a fine balance. Rodent studies showed significant levels of anorexia type tissue damage. More harm than good? Eat sensibly and exercise (both of these are good for you!)! Genome Integrity & Aging In the next few slides we will look at two processes that promote genome integrity - P53 (The Genome Guardian) and Telomere  P53: The guardian of the genome Cellular senescence is a terminal stress activated defence mechanism regulated by p53. Transcription factor- regulates gene expression 50% of all human cancers have a mutation that inactivates p53, usually in it’s DNA binding domain. P53 tetramer complexed with DNA P53- The guardian of the genome Moderate genome damage –induce DNA repair – return to normal Severe genome damage (e.g. telomere dysfunction) – senescence Severe genome damage plus hyperproliferation - apoptosis Cellular senescence as a defence mechanism Cellular senescence is a stable (usually irreversible) cell cycle arrest It is a defence mechanism to protect an organism for potentially dangerous cells (e.g. pre-cancerous). It is a tumour suppressor function. Multiple causes can trigger cellular senescence - response to DNA damage, telomere loss, loss of nuclear integrity and other forms of cellular damage. During ageing it can contribute to depletion of stem cells. Accumulation of senescent cells is a main hallmark of ageing. Also, possibly a major cause of ageing.  Telomeres Telomeres are repetitive DNA sequences at end of chromosomes. DNA polymerase is incapable of replicating all the way to the end of a linear chromosome (eukaryotes). So the ends of chromosomes shorten with every replication cycle – losing telomere repeats. Once all the repeats are gone this shortening continues and deletes genes.  What happens when cells lose telomeres? Can healthy cells taken from tissues grow in vitro indefinitely? Human fibroblasts Foetus- ~60 doublings 80-year-old adult- ~30 doublings What causes this? Clear correlation between organismal life expectancy and number of cell culture doublings The Hayflick limit The Hayflick limit discovered by Leonard Hayflick in 1960s. Cells dividing in cell culture divide finite number of times before they have severe telomere shortening or loss. They have become senescent – induced by P53.  DNA synthesis DNA polymerase can only work in the 5’ to 3’ direction. Needs a “primer” to initiate polymerisation. Initiated by RNA primers –that degrade and leave single stranded DNA The chromosome end problem RNA in blue, newly synthesised DNA in green Single stranded DNA gets degrad Telomerase – an enzyme that grows telomeres Telomerase is composed of two sub-units. Reverse Transcriptase and Telomerase RNA. Reverse Transcriptase 3 repeats New DNA synthesis T A 5'-TTAGGGTTAGGGTTAGGGT 3’-AATCCC 3'-CAAUCCCAAUCCC-5'. Telomerase RNA 4 repeats – etc. Sequential rounds of growth. 5'-TTAGGGTTAGGGTTAGGGTTAGGG 3’-AATCCC 3'-CAAUCCCAAUCCC-5'. This can act as primer for bottom strand DNA synthesis by standard mechanism. 23 Telomerase – grows new telomeres But if chromosomes lose DNA sequence from their ends with every cycle of DNA replication, how can we reproduce? Telomerase – an enzyme that grows new telomerase. Germline stem cells have telomerase and the can grow telomeres. Embryonic stem cells have telomerase – they can be cultured indefinitely. Most adult stem cells have some telomerase, but usually less than germline stem cells. Cancer cells have telomerase. Is ageing of the organism caused by cells reaching Hayflick limit? Few cells reach Hayflick in people but telomere shorten. P53 senses multiple different threats and integrates them to make a decision – keep growing, become senescent or apoptose. Telomere shortening plus other genome integrity defects, accumulations of mutations, and other stresses in combination can trigger senescence. Resulting accumulation of senescent cells and loss of stem cells. Ageing a complex phenotype –but metabolism and genome integrity play a major role.