Biological Clocks and Ageing

Intraterrestrial life extends down at least 5 km and animals are found even in the deepest oceans. The biosphere is, therefore, dominated by dark, largely “arrhythmic” habitats, and in terms of biomass, most of life on earth resides in places isolated from the direct effects of the sun…studies of species that live away from the sun are a very small fraction of rhythmic physiology research. A D Beale 2016.

This posting looks at whether a decoupling between circadian rhythms and the metabolism/redox could be resulting in slow ageing, and in some species reduced cancer rates or improved regeneration capacity.

Difference in circadian rhythms might also influence synchronisation – with biological timers influencing processes throughout the body.

Even species without a ‘traditional clock’ would have 24 hour rhythms through redox oscillations (including of peroxiredoxin), but it is not clear whether this would definitely be the case, or whether the redox oscillations would span more than 24 hours if they were not coupled to a circadian clock system.

During the last decade, an increasing number of studies have appeared that have questioned the free radical theory of the aging process. Conflicting results have been reported in experiments on C. elegans but also on rodents, which clearly reveal that ROS production does not control the aging process. There are long-lived species and genetic mutants displaying a high level of oxidative damage and chronic oxidative stress.

The Naked Mole Rat

The naked mole-rat, the longest-lived rodent with a lifespan over 30 years, is a striking example refuting the age-related oxidative degeneration theory. Naked mole-rats display lower antioxidant capacity, elevated lipid peroxide concentrations and significantly higher levels of oxidative damage to lipids, proteins and DNA than physiologically age-matched mice did. It seems that naked-mole rats can tolerate oxidative stress and damage better than their short-lived counterparts via mechanisms currently unknown. These rats are also protected against tumorigenesis and show a striking decline in the appearance of senescent phenotype although they express clearly elevated levels of ROS and oxidative damages. Differences have also been recently found in the mole rats pluripotent stem cells. S Miyawaki – ‎2016.  Cancer is rare (although not unheard of) in mole rats.

C Elegans

Studies indicating that the increase in oxidative stress, e.g., by knocking out antioxidants, can extend the lifespan of Caenorhabditis elegans (C. elegans). Moreover, superoxide dismutase, an enzyme, which is indispensable for the detoxification of superoxide radicals, is not obligatory to achieve a normal lifespan of C. elegans.  Sanz et al. also conducted many experiments demonstrating that mitochondrial ROS production was not crucial for the control of lifespan in Drosophila A Salminen 2013. 

Both molerats and c.elegans evidence hard to quantify circadian rhythms.

Solitary and social mole rats typically show clear circadian rhythms and entrainment when monitored in a laboratory, suggesting they have a functional circadian clock, but eusocial naked mole rats (Heterocephalus glaber) and Damaraland mole rats show attenuated or no circadian clock rhythms..G Bloch 2013.

And although circadian rhythms have been described in the nematode Caenorhabditis elegans at the behavioral level, these rhythm are not robust. In contrast to other animal models, no circadian transcriptional rhythms have been identified. Thus, whether this organism contains a bona fide circadian clock remains an open question.  C. elegans rarely acquires the kinds of tumours that can be readily observed in other animals…many of the same defects that underlie cancer in humans do cause excessive cell proliferation in C. elegans, it’s just that the effects are modest. the only cells that form genuine tumours in C. elegans are stem cells, and the likely reason why C. elegans doesn’t get other forms of cancer is that, unlike in mammals (and many other animals) there are no adult somatic stem cells that can serve as the origin of a tumour. N V Kirienko (2010)

Other potential examples of where a lack of a robust circadian clock may result in an increased age span

In some species, there can be a ‘prolonged clock’ (which still have the gears of standard circadian clocks, but the clock no long responds to light), no clock, or a jammed clock, etc. Tina Hesman Saey July 2015. 

A lack of a robust circadian clock can offer species benefits, if they do not have to adapt their foraging to daily cycles.

The Proteid Proteus Anguinus

The proteid proteus anguinus, an obligate cave-dweller, showed no apparent daily rhythm of activity or resting metabolic rate. H Hervant 2015. Proteus anguinus, a small cave salamander (weighing 15–20 g), has evolved an extreme life-history strategy with a predicted maximum lifespan of over 100 years, an adult average lifespan of 68.5 years, an age at sexual maturity of 15.6 years and lays, on average, 35 eggs every 12.5 years. Surprisingly, neither its basal metabolism nor antioxidant activities explain why this animal sits as an outlier in the amphibian size/longevity relationship. Y Voituron 2015.   Because it doesn’t extend much energy, its metabolic rate mostly stays at its baseline rates, while most animals’ metabolisms are often running much above their basal rate. Neotenic axolotls such as the proteus anguinus are able to regenerate full appendages unlike their amphibian relatives (A lovita 2015),

The Hydra

Findings indicate that asexual polyps from most hydra species have extraordinarily long lifespans.  Recent sequencing of the Hydra magnipapillata has reveal that it has lost both Clock and Cycle. S Tomcsyk 2015, but still displays a photoperiodic behaviour in response to life cycles.  (Taddei-Ferretti and Musio 2000). Several studies aimed at dissecting the mechanisms that underlie the lack of senescence in Hydra focused on FoxO, an evolutionarily conserved transcription factor.

In bilaterian organisms, FoxO regulates the response to stress, the proliferation of stem cells, and modulates lifespan  In nematodes and fruit flies, the knockdown of FoxO significantly shortens lifespan. In Hydra, FoxO is expressed in stem cells, and appears to respond to stress. Reduction in FoxO levels in the H. vulgaris AEP strain negatively affected the proliferation of stem cells, the speed of the budding process, the growth of Hydra population, and the production of immune peptides.  S Tomcsyk 2015.

Hydra tumors share several characteristics with human tumors. Hydra tumor cells migrate, appear to be resistant to programmed cell death, and have different gene expression patterns than normal Hydracells.  The stem cells, which are programmed for sex differentiation, accumulate in large quantities and are not removed naturally by programmed cell death. Interestingly, these tumors affect only female Hydra polyps and resemble ovarian cancers in humans. T Domazet-Lošo 2014.   There are ovarian rhythmic cycles that may influence this.

The Mexican Blind Cave Fish

The Mexican blind cave fish store up high fat reserves (which would be expected to accelerate ageing), and yet they are a healthy and relatively long lived species. Cavefish have a mutation in MC4R, a gene known to be regulated by leptin (an appetite-suppressing hormone) and insulin in the human brain. Research also shows that although the blind cavefish has a circadian clock when kept in an environment of daily cycles of light and dark, this is repressed in cave environments. The expression level of Period 1 is very low and not oscillating and there is a significantly raised expression of per 2. There is also a higher expression of DNA repair genes, and a greater ability to repair DNA damage. Current results point to an internal timing process in the blind cave fish – perhaps related to the feeding patterns of the blind cave fish. When food does become available to the blind cave fish – perhaps once a year, – the fish are able to eat without limit and store as much fat as they can.

Gymnosperms

Gymnosperms currently hold the longevity record for living organisms [e.g., the Welwitschia mirabilis. It has also been questioned whether circadian rhythms exist in gymnosperms. Data suggests clock gene conservation in gymnosperms, but functional differences in clock function between gymnosperms and other plant taxa, with rapid dampening of circadian rhythms found in Picea abies. S Gyllenstrand 2013. 

Cicadas

Cicadas have a life span of up to 17 years (the longest life span of any insect in North America), this means they follow a much longer clock cycle, but their life is mainly spent underground. Tina Hesman Saey July 2015. 

E-Coli

In a bacterial population, aging and rejuvenation goes on simultaneously, so previously scientists had believed that E.coli did not age. However E. coli divides, a septum forms in the middle of the dividing cell and then the two daughter cells are pinched apart. As the cell wall seals the break, the two daughter cells end up with one “old” end and one newly-formed end. When the two daughters go on to divide, the process is repeated. The original old ends gets passed on from generation to generation. Division is thought to take place around every 30 minutes. CU Rang – ‎2012. E Coli is not thought to have circadian rhythms, although a clock can be transplanted into the bacteria. A H Chen 2015.

In comparison cynaeobacteria has circadian rhythms and these control the bacteria’s rate of cell division (their method of reproduction) in single cells. “These cells have to keep dividing, and the circadian oscillator regulates when they divide” Q Yang 2010.  It would therefore be interesting to compare the cell division/’ageing’ process in the two types of bacteria.  There is some evidence that the process in cynaeobacteria is somewhat different from that of e-coli. D Mandakovic 2016.  

In some cases, animals living above ground have a longer life span, although this is not comparable to the examples given above. The relative long-lived caribou (Rangifer tarandus) has a weak circadian (24hr) clock. Tina Hesman Saey July 2015, although it still has seasonal cycles such as mating and migration dictated by light sensitive melatonin.  The long lived elephant is active both at night and during the day; they are least active during the hottest part of the day (responding to temperature).  Activity also changes according to environmental changes during the year.  Despite having 100 times more cells, elephant cancer mortality is less than 5%, compared to 11-25% in humans. Evolution has duplicated a tumor preventative p53 gene and provided them with increased resistance.

It would be interesting to see relevant data for other long lived species e.g Pinus longaeva, planarian Worms,Taxus brevifolia, Balaena mysticetus (the Bowhead Whale lives a double centenarian lifespan and it weighs a couple of tonnes, yet cancer is a rarity in this species), turritopsis dohrnii, and somniosus microcephalus, as well as data on species that do not have robust circadian rhythms but are not held to be particularly long lived.

So the secret of a long life may involved a lack of a robust connection between a clock and the metabolism, but the exact mechanisms for this remain unclear.

Perhaps by building on the data it will be possible to look at various mechanisms that apply in the case of the above species with very long life spans and understand whether these mechanisms apply in all cases e.g this might include:

  • a ‘prolonged clock’ (which still have the gears of standard circadian clocks, but the clock no long responds to light).
  • Differences in expression level of clock genes e.g significantly raised expression of per 2 and higher expression of DNA repair genes, and a greater ability to repair DNA damage.
  • low body temperature and/or lack of thermoregulation (the naked molerat is cold blooded.)
  • neoteny (including Proteus anguinus, Naked Mole Rat, and the Bowhead Whale)
  • intermittent calorie restriction e.g during during times of drought , molerats are unable to burrow and gather new tuber supplies. The mole rats then have no alternative but to fast. A Lovita 2015.
  • oxygen restriction.
  •  an aberrant melatonin system (mole rat).
  • eye degeneration.
  • Altered pain receptors (mole rat).
  • light independent magnetoreception (e.g proteus anguinus and the naked mole rat – which could have implication for responses to oxidative stress).

It may also be possible to eliminate factors that apply in the case of species that do not have robust circadian rhythms but have an average or relative short life span.  It would also be interesting to look at factors applying to very short lived species.

What might this mean for animals which live above ground and have a strong circadian clock

On the basis of above findings it is speculated that a prolonged circadian clock (combined with other factors) in some species may be involved in extending life and even allow continued stem cell regeneration, because of a weakened coupling of redox and circadian rhythms (perhaps resulting differences in the anti-oxidant cycle, DNA repair, and metabolism).

But it is unlikely that this approach will be that useful for all species, as in those dwelling above ground there may have been an evolutionary need to develop a strong link between circadian rhythms and the metabolism (i.e nutrition, photosynthesis, respiration, etc), responding to more extreme changes in light and temperature, supporting migration, and stress resistance to biotic and abiotic cues.

In species that required robust circadian rhythms, the loss of circadian rhythms during the ageing process may result in damage, as the circadian-redox coupling may be very different. Mouse models without functional circadian clocks sometimes exhibit reduced life expectancy. This may be due to their inability to properly control and synchronize energy expenditure, affecting, for example, the integrity of neurons in the brain. Endogenous circadian clocks orchestrate several metabolic and signaling pathways that are known to modulate lifespan, suggesting clocks as potential targets for manipulation of metabolism and lifespan. S D Katewa 2016.

The following findings may be of more relevance to species with a strong circadian clock.

The Emerging Link Between Circadian Rhythms and the Metabolism/Redox

Cellular reactive oxygen species (ROS) sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism… accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival P D Ray 2012. 

In addition to the well-established role of the mitochondria in energy metabolism, regulation of cell death has recently emerged as a second major function of these organelles. This, in turn, seems to be intimately linked to their role as the major intracellular source of reactive oxygen species (ROS) which are mainly, generated at Complex I and III of the respiratory chain. Excessive ROS production can lead to oxidation of macromolecules and has been implicated in mtDNA mutations, ageing, and cell death.  S Orrenius 2007.

Recent findings strongly suggest that the circadian clockwork is involved in complex cellular programmes that regulate endogenous ROS and also defend the organism against exogenous oxidative challenge. Current evidence seems to support the conclusion that the responses to ROS are mediated both through the regular function of the molecular clockwork and the involvement of the TTFL (transcription–translation feedback loops producing oscillations with a period of approximately 24 hours) genes in extra-circadian pathways. Lisa Wulund 2015Nishio 2015, N B Milev 2015.  A Stangherlin 2013.

Redox signalling comprises the biology of molecular signal transduction mediated by reactive oxygen (or nitrogen) species. By specific and reversible oxidation of redox-sensitive cysteines, many biological processes sense and respond to signals from the intracellular redox environment. Redox signals are therefore important regulators of cellular homeostasis. Recently, it has become apparent that the cellular redox state oscillates in vivo and in vitro, with a period of about one day (circadian). M Putker 2016. Oxidation–reduction cycles of peroxiredoxin proteins are thought to constitute a universal marker for circadian rhythms in all domains of life. R S Edgar 2012. They may not be unique in their ability to undergo redox oscillations since many other proteins are susceptible to oxidation of their cysteine residues by peroxide. S Ray 2016.

There are a number of examples of how this coupling between redox and circadian operates in practice e.g

  • The Cryptochrome protein (CRY) in Arabidopsis, Drosophila, and mouse provide the most direct path by which redox status can interact with the core components of the transcription–translation feedback loop (TTFL). CRY shares homology with a phylogenetically ancient enzyme family that repairs DNA in response to ultraviolent light known as the photolyases, leading to speculation that redox is therefore crucial to CRY function. Indeed, the CRY protein does contain motifs that bind the flavins, a group of organic compounds known for their prominent role in electron transport in many metabolic reactions and also their ability to be reduced by the incidence of light. Lisa Wulund 2015.   Also see M L Fanjul-Moles 2015. 
  • A protein disulfide isomerase (PDI) from Chlamydomonas reinhardtii (CrPDI2) is involved in the circadian signaling pathway and, together with the night phase-specific interaction of CrPDI2 and a peroxiredoxin, findings suggest a close coupling of redox processes and the circadian clock in C. reinhardtii. A Filonova 2013 

The coupling may also be found in SIRT 1.   Sirtuin 1 (SIRT1) is involved in both aging and circadian-clock regulation, yet the link between the two processes in relation to SIRT1 function is not clear. Using Sirt1-deficient mice, it has been found that Sirt1 and Period 2 (Per2) constitute a reciprocal negative regulation loop that plays important roles in modulating hepatic circadian rhythmicity and aging. R H Wang 2016.

This enzyme can regulate several survival functions by deacetylating not only histones but also many crucial transcription factors e.g., those controlling autophagy and ROS production. SIRT1 can stimulate the expression of antioxidants via the FoxO pathways. Moreover, in contrast to ROS, SIRT1 inhibits NF-κB signaling which is a major inducer of inflammatory responses…Recent studies have demonstrated that an increased level of ROS can both directly and indirectly control the activity of SIRT1 enzyme. For instance, ROS can inhibit SIRT1 activity by evoking oxidative modifications on its cysteine residues. Decreased activity of SIRT1 enhances the NF-κB signaling, which supports inflammatory responses. This crosstalk between the SIRT1 and ROS signaling provokes in a context-dependent manner a decline in autophagy and a low-grade inflammatory phenotype, both being common hallmarks of ageing. A Salminen – ‎2013

The Link Between Circadian Rhythms and Morphogenesis

Circadian rhythms permeate mammalian cell biology, with ‘omics studies in tissues and isolated cells both revealing that significant proportions of gene expression and metabolism are circadian regulated  with a commensurate impact upon biological function. A recent report by  found that more than a third of protein-coding genes in the mouse genome exhibit circadian regulation in one or more tissues. In consequence, it should not be surprising that from cell division to signal transduction from inflammation to neuronal long-term potentiation circadian rhythms modulate cellular activity to support anticipated demand. M Putker 2016.

Increasingly evident is that metabolic homeostasis at the systems level relies on accurate and collaborative circadian timing within individual cells and tissues of the body. Eckel-Mahan 2013, J S O’Neill 2014, JD Johnston – ‎2016. In animals, the current understanding of cellular circadian rhythms throughout an organism is that while the core clock genes are oscillating in most tissues and in the midst of enormous environmental pressures, metabolic circadian oscillations are strongly shaped by the environment…feeding is a critical modulator of the internal clock and, in addition to affecting synchronicity between the central pacemaker and metabolically active peripheral tissues, it likely controls the extent to which peripheral tissues are in phase with one another.  A Ribas-Latre 2016

S A Brown (2014) has provided an overview of the molecular mechanisms involved in circadian clocks and then discuss how such mechanisms can influence stem cell biology and hence tissue development, homeostasis and regeneration. Cell differentiation, e.g. that of stem cell populations, might be also under circadian influence. Studies have shown that clock genes can indeed directly influence stem and progenitor cell fate. S Brown 2014,  Y Inada 2014 X Yu 2013. Circadian clock genes have recently been found to modulate human bone marrow mesenchymal stem cell differentiation, migration, and cell cycle. H Boucher 2016.  With age, a reduction of stem cells is observed, which may affect the maintenance of tissue function. Previously, it was demonstrated that the circadian clock affects the equilibrium between self-renewal and differentiation of epidermal stem cells.  S S. Fonseca Costa 2015

A number of studies have suggested strong links between circadian rhythms and cancer. S Sahar 2009, Antoch et al., 2013Fu et al., 2002,Cao et al., 2009Yang et al., 2009), C H Johnson CH 2010,XM Tan – ‎2015, R V Puram 2016. 

  • In rodent studies exploring how cancer in one organ spreads to others, it was found that lung adenocarcinoma sends signals to the liver through an inflammatory response, which rewires the circadian mechanisms that manage metabolic pathways. As a result of this inflammation, the insulin signaling pathway is inhibited in the liver, leading to decreased glucose tolerance and reorganization of lipid metabolism. Lung tumors take control of circadian metabolic function in the liver, potentially to support the heightened metabolic demands of cancer cells. It is believed that this distal rewiring of metabolic tissues does not occur only in the liver, suggesting a systemic shake-up of metabolism. Sassone-Corsi 2016.   There have been similar findings for colorectal cancer. SA. Huisman 2016, and breast cancer NH Ha – ‎2016.
  • In recent studies in animal  models it has been found that leukemia stems cells have the capacity to self-renew and propagate disease upon serial transplation.  Both normal and malignant hematopoietic cell harbour an intact clock with robust circadian oscillators and genetic knockout models reveal a leukemia specific dependency on the pathway.  These findings establish a role for the core circadian clock genes in acute myeloid leukemia.  R V Puram 2016.
  • Studies of mice with disrupted circadian rhythms and sleeping patterns (compared to controls) have been found to gain weight, develop fatty livers and be more susceptible to liver cancer. N M Kettner 2016 
  • The enzyme TERT and its activity were found to be under circadian control in mice. Circadian expression of TERT mRNA is hardwired to the circadian oscillator via direct regulation by the BMAL1 and CLOCK heterodimer. Mice deficient for CLOCK do not display rhythmic telomerase activity and their chromosomes have shorter telomeres. On the other hand, it was shown that reconstitution of TERT into senescent fibroblasts could reconstitute their circadian system. Taken together, the interplay between TERT and the circadian oscillator may contribute to aging. S S. Fonseca Costa 2015. W D Chen 2014. Telomerase activity has been found in almost all human tumors but not in adjacent normal cells. The most prominent hypothesis is that maintenance of telomere stability is required for the long-term proliferation of tumors. J W Shay 2001.

Conclusion

Increasing our understanding in species with weak circadian rhythms, and growing evidence of the importance of the redox-circadian coupling in species living above ground (subject to the daily cycle) may throw further light on this subject in the future.

October 2016. This article merely joins up other peoples work into an overall system.  These works have been referenced so it is clear that others have provided the individual pieces of evidence that have been used to shape a specific systems approach.   

 

 

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