BIOL 364: Biology of ageing - Lecture 05 - Mitochondria and ageing, Telomeres and ageing

Learning Objectives

Be able to;

Evaluate the theory and evidence for mitochondrial damage and dysfunction as a significant cause of ageing.

Explain the ways in which mitochondria may be involved in signalling with the nucleus to modulate ageing.

Describe the evolution of telomeres and explain why telomere erosion is more likely to be a consequence than a cause of ageing.

Describe how cell senescence can occur, and why it exists, and argue whether it contributes to ageing.

Semi focus - We will explore the idea of secondary damage and causes of ageing, instead of the primary causes of ageing we investigated like oxidative damage and AGEs.

What mitochondria really look like

Not just sausages inside cells - they are like that in muscle cells (cardiac and skeletal) as they are packed with mitochondria for energetic requirements.

Nerve cells also require a lot of energy and mitochondria can run the length of an axon in the cell.

Mitochondria can also form a trabecular network in some cells; mitochondrial DNA rings a nucleus with networks forming around it.

This is not that important, just that mitochondria are more complicated than we often give them credit for.

Iborra et al. 2004 - mitochondrial network formed around the nucleus.

What is the Role of Mitochondria within the molecular damage model of ageing?

Cascade process; Insults & metabolism (mitochondrial activity - causes damage due to ROS? As ROS forms almost exclusively in the mitochondria due to oxidative phosphorylation) -> Molecular damage accumulation -> Cellular damage / cellular dysfunction -> Cell death & Cancer

Leads to;

Tissue/organ dysfunction -> Organ failure -> Systemic failure -> It ends in Death

Why is Mitochondria involved, causally, in Ageing?

Mitochondrial damage and ageing: A secondary mechanism?

Mass-specific metabolic rate correlates inversely with lifespan in mammals (generally - although there is a bit of spread around the correlation line);

Metabolism produces ROS;
ROS lead to molecular damage with age that can accumulate (DNA, proteins, lipids, AGEs) despite detox/repair systems that we have (FRT);

Observationally, Longer-lived species produce less ROS/O2 (less ROS per O2 consumed) used (generally, with some exceptions).

ROS nearly all produced by mitochondria.

mtDNA mutation rate 100-1000X higher than nucleus - mitochondrial mutation rate is much higher - no proofreading or repair enzymes in the mitochondrial DNA compared to the nucleus.

The likelihood of mt membrane lipid peroxidation correlates inversely with lifespan (membrane pacemaker theory) - membrane peroxidability is not really genetically controlled within a species - we can't change our membrane composition and therefore, how peroxidiaable it is.

So mitochondrial damage might contribute to differences in lifespan between species...

How about within species? (what causes ageing within a species?)

What causes ageing within a species?

Mitochondrial damage and ageing – within species.

Mitochondrial function declines with age.

Low age-related increase in mtDNA damage (not massive but it's apparent).

We know there is an Age-related increase in protein ox (oxidative) damage? → No clear evidence due to increased mtDNA mutations → No vicious cycle as not supported by evidence (mt damage & mutation → bad mt protein → more ROS) - FRT was modified in the 70s? - to mt FRT (mitochondrial Free Radical Theory of Ageing) - an attractive theory, but not supported by evidence.

What we do see for mitochondrial causes of Ageing;

Lon protease (a mt matrix protein that degrades misshapen proteins and is part of proteostasis machinery in mt) declines with age in sedentary (less active) mice compared to more active mice.

A good review that followed from this study (link here) - 12 hallmarks of Ageing, mitochondria is there as one of the causative factors.

Papers following this study were interesting; The role of mitochondria in ageing is a big area of interest (sensible reviews here: 1, 2), and the Mitochondrial FRT of ageing endures (more reviews here: 1, 2, 3, 4).

There are objections though.

Despite strong objections (links: 1, 2, 3) - to it having a major or contributing role.

Emerging area: mt protein homeostasis. - over the past 5-8 years.

PolgA (a mt polymerase) – the mt mutator mouse story

Mice engineered with proof-reading deficient PolgA (catalytic subunit of mtDNA polymerase).

Rationale: PolgA mutant (a mitochondrial polymerase that lacks proofreading) → more mtDNA mutations → fast ageing.

Observations: A phenotype that looked a lot like ageing;

increased pathologies (osteoporosis, hair loss, hair greying, weight loss, cardiomyopathy, hearing loss, and low fertility).

A much shorter lifespan.

Is it premature ageing?

We should be cautious about things called accelerating ageing - an individual that just looks old and dies young - should be suspicious if that is actual ageing.

It seems to be ageing.

It could be akin to progeria.

Follow-up: do point mutations limit PolgA mutant lifespan?

Both homozygous and heterozygous mutants have more point mutations in energetically demanding tissues like the heart and the brain.

Several hundred more mutations in the old wild type than young will type, with even more in the homozygote.

The problem of the paper;

But: look at lifespan vs point mutations - However, the point mutations did not reflect the lifespans that were measured.

Whilst the homozygote PolgA mutant had the expected greatly shortened lifespan, but the heterozygote lived around the same length as the wild type despite having many more mt mutations.

Why is that?

Why do the homozygote PolgA mutant have the expected greatly shortened lifespan, but the heterozygote lived around the same length as the wild type despite having many more mt mutations?

A follow-up research paper from a different group; mt deletions, not point mutations limit PolgA mutant lifespan.

Heterozygous vs WT: similar deletion rate.

Heterozygous vs WT: similar lifespan.

By looking at mt deletions and not point mutations, they found that the accumulation of deletions with age is what resulted in the reduction in lifespan - they found that the WT and the heterozygote group had a much lower rate of deletion mutations than the homozygote with many more deletions with age.

So, do mtDNA deletions cause ageing?

It’s quite easy to shorten lifespan. - when we look at something that shortens - its not good to immediately associate it with causing ageing - we need more evidence for that as it is very easy to shorten the lifespan of organisms and it is not causative proof of ageing.

Good evidence, if possible, would be if longer-lived flies had fewer deletions, or if deletions were reduced, the lifespan increased.

What are the number of stages mitochondria (Mitochondrial proteostatsis) go through depending on the severity of the insult of damage done to them?

Molecular QC - when damage is mild, the chaperones and proteases can manage it - misfolding response and proteins will be turned over (by Lon Protease and others) - e.g. reduced Lon protease.

More severe damage (but not to the limit of damage that will kill it); Organellar QC.

Stress-induced mt hyperfusion;

Mitochondrial fusion due to substantial damage - membranes fuse and content mixes - you get a reduction in the level of damage and malfunction in the mitochondria suffering the damage.

MIchondrial fission; Too much damage; (cf. Lysosomes, lipofuscin) - A signal is sent to snip off that bit of the mitochondria (fission) - that bit that was snipped and damaged, signals are sent to segregate it for mitophagy (see it in Lysosomes & Lipofuscin).

Cellular QC; if the damage is very severe - never going to be a single bit of a mitochondria or single mitochondria experiencing that damage - that damage is experienced by the whole cell.

The mitochondria signal for cell death through the comprised mt outer membrane permeability and cytochrome C is released, which signals for the whole cell to undergo apoptosis.

Hormesis is the idea that a sublethal thing that might be considered detrimental can lead to an increased health advantage later.

Hormesis formalised in 1943 - first idea Virchow 1854: tracheal cilia beat better at low doses of NaOH and KOH (strong oxidants).

Link to 100 BC Pontus king Mithridates: to immunise himself from poisons - took small amounts to immunise himself from poisoning - negative stimulus cannot be too great or else it becomes detrimental (his father died poisoned), he was taking multiple poisons regularly at small doses.

With hormesis, the negative stimulus cannot be too great, otherwise, it would become permanently harmful.

Hormesis example, a small heat shock to fruit flies early on in their life can significantly extend their lifespan by about 5%.

Another Hormesis e.g; Intermittent starvation.

Mitohormesis: mild mitochondrial stress promoting cellular health.

Retrograde response, electron transport chain disruption, mild metabolic stress (e.g. exercise, DR) signals stress (can induce hormesis).

Hormetic effects are modulated by the Nucleus: upregulation of stress-coping mechanisms.

Promotes genome stability, energy mobilisation (fatty acid (FA) beta-oxidation), autophagy & mitophagy upregulation, TOR inhibition.

A dial, not a switch - not an on or off thing - if you turn it too far you would hurt yourself.

Maybe blocked by antioxidants! - exercise produces these signals for hormesis that are from ROS byproducts - antioxidant by-products stop that signal from getting to the nucleus for upregulation and prevent all the good things from coming down the pathway.

Not only at the mitochondrial level (cellular level too).

Model: mitochondrial role in ageing - mitochondria are a big conduit for this process as they produce ROS and can produce more under great energetic demand.

ROS can have detrimental and beneficial effects here, leading to increased or reduced damage.

+ and - effects for mitochondria on ageing, including Mitohormesis, apoptosis, oxidative damage, energy depletion (may lead to necrosis)
Too much ROS can cause dysfunction.

But low levels of stress cause only transient dysfunction and can do retrograde signalling can produce stuff like 4HNE - a stress signaller.

Which can upregulate survival and maintenance mechanisms, through the nucleus.

A theory supported by evidence; mitochondria might be an example of antagonistic pleiotropy (APTA).

Rapidly growing cells are limited by ATP production;

These cells thus actively inhibit mitophagy, if the mitochondria can still function, to maximise mitochondrial ATP production and compete successfully for scarce nutrients;

This process maximizes early growth and reproduction, but permits the persistence of damaged mitochondria.”

However it also;

Increases mtDNA mutations and deletions when replication depletion occurs or replication is not performed as it should.

When you get damage to PolgA, you get an increase in mitochondrial mutation deletions and may be at the bottom of this.

The mechanism of the theory is that ROS can inhibit insulin dampeners and allow insulin-like signalling (ILS); increase lifespan and be good for growth.

Mechanism invoked: inhibition of mitophagy via ROS‐dependent activation of insulin signalling (IIS) - growth pathway including Tor - not totally clear but a little bit of evidence for this (not unreasonable).

Short-term benefit, long-term issue: Antagonistic pleiotropy.

Mitochondria -ageing review

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779179/

Do Telomeres cause ageing?

NO!

What do Telomeres do;

Protect against uneven chromosome segregation & cancer, as when not eroded they prevent the ends of chromosomes from joining each other.

Chromosomes lose telomeric DNA with each cell division.

Cell cycling arrests when telomeres eroded (Hayflick limit).

What is the The Hayflick limit?

A limit to the number of divisions that any given cell (most) can undergo before there is a cell cycle arrest.

Cellular senescence (protective mechanism against cancer here)

If a cancer cell keeps dividing it can invade the whole body - if there is a limit to the number of times it can divide and it ends up being arrested and entering replicative senescence - then it is no longer a cancer cell, but...

One-way cancer cells can evade cell (replicative) senescence and keep dividing is by activating telomerase.

Why we think that telomeres are probably not terribly important in lifespan determination

The initial argument was a stem cell depletion argument: telomeres erode, stem cells senesce, no rejuvenation.

we lose cells to damage, so we need pools of stem cells to renew new cells.

BUT;

Mice have constitutively active telomerase → long telomeres (have telomerase constitutively expressed too).

Humans have repressed telomerase → short telomere (our telomerase is repressed).

Mice don’t get stem cell depletion but humans do.

Both get old and die.

Yet humans live way longer (80 years vs 3 years - so it ain’t the telomeres).

Telomere shortening and replicative cellular senescence are unlikely to be a general cause of ageing.

But why the big difference in telomeres? (and telomerase).

What is the Theory of Short telomeres, replicative senescence and cancer suppression?

Theory;

Evolution of homeothermy -> an enormously increased metabolic rate (a higher energetic requirement) -> more ROS production -> more DNA damage -> more telomere erosion -> More Cancer.

The idea is that Replicative senescence evolved against carrying ‘bad cells’ (fused chromosomes or cancer cells).

Replicative Senescence evolved organisms didn't carry bad cells, which started appearing because of more free radicals.

Supportive Facts:

Replicative cell senescence protects against tumour progression.

85-90% of malignant biopsies are telomerase +ve; They need telomerase so that they can keep dividing.

Gomes, et al. (2011), Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination. Aging Cell, 10: 761

Evolution of Telomeres

Evolutionary facts;

The mammalian ancestor had the human type: short telomeres and repressed telomerase.

Aquatic poikilotherms (ancestors of mammals, which appeared earlier during evolution) had short telomeres & expressed telomerase.

It’s likely that telomerase repression evolved to protect against tumours - for replicative cell senescence to avoid tumour formation (perhaps, unable to exactly tell).

Mouse type (long telomeres, constitutive telomerase) then evolved multiple times independently.

Why then?

Gomes, et al. (2011), Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination. Aging Cell, 10: 761

Why did mice evolve independently many times?

Possibly Trade-offs (Theory);

Costly, in fitness terms, to evolve and maintain resistance to oxidative damage (is it Easier to evolve long telomeres and to switch on telomerase, than it is to evolve a lot of ton of antioxidant mechanisms and get the genetic machinery to upregulate and coordinate those things - more difficult to do).

It might also be more costly to forego regenerative capacity (stem cells), to heal yourself.

Especially for short-lived animals that endure lots of damage.

So it might have been easier to evolve longer telomeres to cope with erosion.

Telomeres, oxidants and lifespan.

That Theory suggests, with Evidence through correlations across species, that;

Longer-lived organisms should have; Shorter telomeres & repressed telomerase.

Body mass is strongly correlated with lifespan in mammals.

Shorter telomeres -> More resistant to oxidants - less statistically clear - fewer data points.

Shorter-lived organisms;

Telomerase is not correlated with lifespan but is negatively correlated with lifespan

Longer telomeres = shorter lifespan (don't have to be that resistant to oxidation? Not as much evidence and is less clear).

Species with high extrinsic mortality may have reverted to long telomeres to limit somatic maintenance costs and boost cell stemness, to cope with injuries.

Especially if they are preyed upon and commonly need to escape predators.

Gomes, et al. (2011), Comparative biology of mammalian telomeres: hypotheses on ancestral states and the roles of telomeres in longevity determination. Aging Cell, 10: 761

What about Telomeres in humans?

Short telomeres associated with CVD (cardiovascular disease).

Observations suggest that humans with longer telomeres have fewer hazards over time.

It Does not mean causative (cf. grey hair)!

However;

“In the West of Scotland Coronary Prevention Study (WOSCOP) trial (Brouilette et al., 2007), pravastatin treatment attenuated the risk of coronary heart disease even though the shorter telomeres persisted, illustrating that the short telomere is not causatively linked to coronary artery disease.”

Can affect CVD risk through a statin, which does not work through a telomere length mechanism, which suggests telomere length itself is not causally linked to CVD risk.

Telomeres in humans?

There is no correlation/association between telomere length and frailty in male and female humans (grip strength, balance & others).

Frailty index and Telomere Length (a) male and (b) female (Woo et al., 2008).

Telomeres and ageing? No. Telomeres and cancer

They were not interested in telomeres and ageing - but in using telomerase inhibitors to treat cancer.

Imetelstat – a telomerase inhibitor against multiple cancers.

Extra reading;

Does telomere length predict lifespan between strains of mouse?

Is Telomere Length a Biomarker of Aging? A Review

BUT …. Lifespan-extending telomerase gene therapy in mice!!

An overexpression of telomerase which leads to a fairly substantial lifespan increase.

Controls are a bit short but 40 months is a pretty good increase compared to normal mouse lifespan.

So perhaps telomerase is linked to ageing - who knows? (as mice are supposed to have telomerase anyway!)

Treatment once/month starting at 18m.

https://www.biorxiv.org/content/10.1101/2021.06.26.449305v1.full

Does Cell senescence contribute to ageing causally?

It is widely acknowledged that Cell senescence contributes casually to ageing.

Cell senescence – what is it?

Stress- or damage-induced.

irreversible, non-dividing state.

not cell death; cells still metabolize & still express genes.

cells tend to increase in size.

express p16INK4a (cyclin-dependent kinase inhibitor – tumour suppression).

express senescence-associated β-galactosidase (SA- β-gal) – increased lysosomal mass.

Initially observed in fibroblasts.

Possible ageing mechanism due to depletion of stem and progenitor cells (maybe in humans not in mice)?

Spotting senescent cells

Mouse embryonic fibroblasts;

2nd (early) passage.

8th (late) passage - cell becomes much bigger & fatter.

Senescence-associated β-galactosidase (SA- β-gal).

Cell senescence – causes;

Causes;

Telomere erosion.

Oncogene overexpression will cause G1 arrest if there is too much of it.

DNA damage due to ROS.

Mitochondrial dysfunction if the damage is too great but not great enough for apoptosis can induce cell senescence.

Inflammation can also induce cell senescence.

Why is cell senescence possibly a cause of ageing?

Due to SASP – senescence-associated secretory phenotype;

SASP varies between cell types but proinflammatory cytokines most common, e.g. GM-CSF (Granulocyte-macrophage colony stimulating factor) & others.

Oher inflammatory factors; produced locally & chronically, possibly enter systemic circulation in sufficient amounts, it is thought that the known state, inflammatory state, of older age and higher inflammatory markers (inflammaging?), is contributed to by senescent cells.

interleukins IL-6 and IL-8 are produced -> leads to inflammation, and the stuff they secrete supports epithelial-mesenchymal transition (which is what a cells needs to becomes metastasis-capable cell).

So if you have cells with growth dysregulation in the local area or systemically, senescent cells can support their transition to becoming metastatic.

So inflammatory supports cancer.

MMPs – matrix metalloproteinases (remoldeling enzymes which can be produced locally) -> collagen loss?

GROs – growth-regulated oncogene proteins -> promotes cell proliferation.

VEGF -> promotes angiogenesis.

These things are great for cancer.

But we have evolved it for wound healing.

So some factors have (physiol) context-dependent effects.

Why is SASP (senescence-associated secretory phenotype) a perfect example of antagonistic pleiotrophy (APTA)?

With a wound you get cell damage; A number of cells will enter a senescent state and secrete SASP which is good for healing, but not so good if you live long.

Angiogenesis.

Cell proliferation.

Chemotherapy resistance.

Epithelial-to-mesenchymal transition.

Stem cell renewal and differentiation.

Inflammation.

Tissue repair.

Recommended read!

Cell senescence – why? - Summarisation of previous points.

Beneficial short-term effect: stops division of damaged cells, eventually kills them → prevents cancer (M. Abad, et al. Reprogramming in vivo produces teratomas and iPS cells with totipotency features. Nature, 502 (2013), pp. 340-345).

also, good evidence for positive role in tissue repair by resolving fibrosis (e.g. MMPs). Wound healing.

Detrimental chronic effect: generates a noxious cell microenvironment, disrupts tissue integrity, promotes chronic inflammation → promotes cancer.

But cancer is a disease of ageing, and so SASP might escape selection…

example of antagonistic pleiotropy.

Are Known disease associations with cell senescence age related?

Known disease associations with cell senescence - a number of which are age-related.

Senolytics, designed to get rid or dampen down the effects of senescent cells.

Cell senescence is involved in these conditions.

A Transgenetic experiment in mice; Killing senescent cells improved health and lifespan (not max LS).

Transgene, INK-ATTAC, induces apoptosis in p16Ink4a-expressing cells of wild-type mice by injection of AP20187, from 12months old.

Not a maximum lifespan extension but a substantial median life span extension - that’s what we like - an intervention has ameliorated what may have been causing ageing for a lifespan extension.

Recommended reading;

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature16932.html

http://genesdev.cshlp.org/content/33/3-4/127.long

Senolytics remove senescent cells in humans.

Dasatinib + quercetin, 9 subjects, diabetic kidney disease patients, avg age 68.7y. Single 3d oral treatment.

With a single treatment over three days, we saw big changes in adipose tissue;

P16+ cells decreased substantially.

SA-Bgal+ (senescent cells) decreased substantially.

Macrophages, which produce GM-CSF, were also reduced a great deal in the adipose tissue.

SASP gene expressions reduced; IL-1,6,9, MMPs, FGF-2 & GM-CSF.

https://www.sciencedirect.com/science/article/pii/S2352396419305912?via%3Dihub

Why do milieu, not stem cells, limit muscle regeneration in old mice?

Is it defunct stem cells that limit regeneration?

Or is it the Milieu in which the cells need to work (bloodstream/lymphatics) in older animals which causes this impaired regeneration?

They were testing if; with dry ice applied on the gastrocnemius of the mouse, would the old mice get impaired muscle regeneration but young mice regenerate their muscles fine?

They get mice, slice them down the side to join up their circulations and stitch them back together to generate parabiotic mice.

Young Isochronic;

Both young mice.

Good regeneration of muscle.

Old Isochronic;

Both old mice.

Impaired muscle regeneration.

Heterochronic;

Old & young mouse parabiotic pair.

Good muscle regeneration.

So; if the stem cells were the problem, which was in situ, the muscles would not have regenerated well.

This shows that it is the stuff surrounding the stem cells that affects muscle regeneration.

Blood biomarkers of ageing & vampire stories

Parabiosis between young and old mice can help old mice improve brain and muscle function:

https://www.ncbi.nlm.nih.gov/pubmed/24797482

https://www.ncbi.nlm.nih.gov/pubmed/24797481

https://www.ncbi.nlm.nih.gov/pubmed/24793238

Human blood biomarkers of mortality risk:

Article (4 biomarkers: orosomucoid, albumin, VLDL size, citrate):

https://journals.plos.org/plosmedicine/article?id=10.1371/journal.pmed.1001606

Review:

https://www.ncbi.nlm.nih.gov/pubmed/26039142

Younger or cleansed blood transfusions into old people to live longer and healthier? - a good chance that it helps.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662775/, Young Blood Rejuvenates Old Bodies: A Call for Reflection when Moving from Mice to Men, https://www.karger.com/?DOI=10.1159/000492573

What are our Conclusions?

Conclusions;

All the theories we have seen in the past 4 lectures have their detractors, who are still refining them, to reconcile their beliefs with the accumulated experimental evidence.

New formulations of theories of ageing are constantly proposed - no clear answer, but we know about contributors.

Because ageing has not evolved, there may never be a fully unifying theory across organism that explains what causes ageing, but the APTA framework has proved fairly robust so far.

In practice, whether underlying primary causes are known, secondary mechanisms (mitochondria, senescent cells) are fair targets for human health interventions.