(previous session)

At the end of the meeting, Martin Brand and Stuart Kim led a group discussion about the free radical theory of aging. Martin began the discussion by pointing out that “after 50 years, you would expect a theory to accumulate enough evidence to convince us that it’s true or false – but the fact that we’re still discussing it today means that hasn’t happened.” I’m paraphrasing slightly, but that’s the general idea.

Martin Brand (who doesn’t, by the way, adhere to this theory) started by summarizing the evidence in favor of FRTA:

  • “50 million Frenchmen can’t be wrong” (i.e., there are lots of correlative experiments)
  • SOD2 knockout is bad
  • catalase overexpression is good

Stuart rejoined with some contradicting evidence:

  • Superoxide dismutase protects against oxidative stress but has little effect on lifespan in mice
  • Deletion of mitochondrial SOD extends lifespan in C. elegans
  • High oxidative damage levels in the longest-living rodent, the naked mole-rat.

To the last of which, others answered:

  • The naked mole rat isn’t suffering from a global increase in oxidative damage – rather, there are a small number of proteins with increased damage, which may represent antioxidant proteins protecting the rest of the cell
  • There’s no evidence that naked mole rats increase damage with age, which is a more relevant metric

The first two pieces of Stuart’s contradicting evidence were more difficult to challenge. Some ideas:

  • Overexpressing an antioxidant enzyme in the wrong subcellular compartment wouldn’t be predicted to have any effect on lifespan

Martin also asked questions about whether FRTA is even falsifiable, and lamented the absence of an alternative clear, single-sentence “singular” theory of aging.

No final resolution but on the balance it seems like the theory is on the ropes, as we’ve discussed here before.


Longevity is correlated with stress resistance. This makes abundant sense: Aging is (among other things) the decreasing ability to maintain cellular homeostasis over time. Cellular stress responses, broadly speaking, detect suboptimal conditions and activation of gene expression aimed at correcting the problem — a fairly reasonably definition of maintaining homeostasis. So it seems reasonable that more robust stress responses (or high basal expression of stress response target genes) would be associated with delayed aging and extended lifespan; indeed, this relationship has been used more than once to screen for long-lived mutants.

The heat shock proteins (HSPs), originally characterized (and therefore named) in the context of cellular responses to high-temperature stress, appear to play a critical role in regulation of lifespan, as illustrated by two examples from C. elegans: Expression levels of hsp-16.2, which vary stochastically even within clonal populations, are correlated with lifespan. Consistent with this, heat shock factor (HSF), the transcription factor that governs the heat shock response, is required for the lifespan extension caused by daf-2 mutations. (Mechanistically, HSF may serve this function by activating target genes that drive disaggregation and degradation of malfolded proteins.)

The heat shock response has now been connected to another major player in lifespan regulation: SIRT1, the most well-studied member of the sirtuin family. In mammals, HSF is subject to acetylation, which diminishes its ability to bind DNA and activate transcription – but this modification can be removed by the longevity assurance factor SIRT1, which is a protein deacetylase. From Westerheide et al. (see also the Perspectives piece in the same issue of Science):

Stress-Inducible Regulation of Heat Shock Factor 1 by the Deacetylase SIRT1

Heat shock factor 1 (HSF1) is essential for protecting cells from protein-damaging stress associated with misfolded proteins and regulates the insulin-signaling pathway and aging. Here, we show that human HSF1 is inducibly acetylated at a critical residue that negatively regulates DNA binding activity. Activation of the deacetylase and longevity factor SIRT1 prolonged HSF1 binding to the heat shock promoter Hsp70 by maintaining HSF1 in a deacetylated, DNA–binding competent state. Conversely, down-regulation of SIRT1 accelerated the attenuation of the heat shock response (HSR) and release of HSF1 from its cognate promoter elements. These results provide a mechanistic basis for the requirement of HSF1 in the regulation of life span and establish a role for SIRT1 in protein homeostasis and the HSR.

The relationship between a master regulator of aging (SIRT1) and an effector pathway (HSF and its target genes) is another example of an emerging trend in the biogerontological literature: the unification of separate longevity control mechanisms. (By “unification”, I don’t mean that these separate mechanisms are shown to be literally equivalent; I simply mean that our increasing knowledge of the connections between genes and their functions has revealed that many phenomena previously thought to act independently are in fact coordinated by regulatory factors).

These findings may also give mechanistic insight into a curious observation from a couple of years ago: resveratrol, an activator of SIRT1, induces the heat shock response. (When we discussed that study, I lamented that the authors hadn’t determined whether SIRT1 was required for the effect — in light of this paper, it does seem that they missed a pretty big boat.) It now seems reasonable to explain those data as follows: resveratrol activates SIRT1, which deacetylates HSF, which in turn binds DNA more efficiently and increases transcription of heat shock response genes. A strong prediction of this model is that HSF should be necessary for any lifespan extension resulting from resveratrol treatment.

We all know people, be they friends, family or colleagues, who seem to have a permanently downbeat view of life. Worryingly for them, research over the last few years has indicated that a negative outlook for the future has been associated with both faster progression of age-related disease, and earlier mortality. Furthermore, telomere attrition occurs with age, and being at the wrong end of the telomere length distribution at any given age is associated with increased risk of a number of diseases, including cardiovascular disease.

So it should come as no surprise to learn that chronic psychological stress has been associated with accelerated telomere shortening in circulating white blood cells. The problem arises in trying to provide a biological mechanism that links this form of stress to increased telomere loss. To address this, a group including the telomere/stress heavyweights Epel and Blackburn noted that both acute and chronic stress have been associated with increased interleukin-6 (an inflammatory cytokine), and repeated bouts of inflammation are associated with telomere shortening. Thus the authors set out to link stress (in this study the measure was optimism vs. pessimism) to telomere shortening via IL-6 in a cohort of post-menopausal women:

Pessimism correlates with leukocyte telomere shortness and elevated interleukin-6 in post-menopausal women.

The combination of less positive and more negative expectations for the future (i.e., lower optimism and higher pessimism) increases risk for disease and early mortality. We tested the possibility that expectancies might influence health outcomes by altering the rate of biological aging, specifically of the immune system (immunosenescence). However, no studies to date have examined associations between optimism or pessimism and indicators of immunosenescence such as leukocyte telomere length (TL) and interleukin-6 (IL-6) levels. We investigated whether dispositional tendencies towards optimism and pessimism were associated with TL and IL-6 in a sample of 36 healthy post-menopausal women. Multiple regression analyses where optimism and pessimism were entered simultaneously, and chronological age and caregiver status was controlled, indicated that pessimism was independently associated with shorter TL (beta=-.68, p=.001) and higher IL-6 concentrations (beta=.50, p=.02). In contrast, optimism was not independently associated with either measure of immunosenescence. These findings suggest that dispositional pessimism may increase IL-6 and accelerate rate of telomere shortening. Mechanistic causal relationships between these parameters need to be investigated.

As with the previous Epel/Blackburn paper (showing that “intrusive thoughts” impact on telomere length), the psychological measures (optimism, pessimism and perceived stress) were all based on questionnaires. As a molecular biologist who is accustomed to running standardized assays I will always have a little trouble when the data comes from a subject assigning a score to a comment such as, “If something can go wrong for me, it will”. I worry that there is such a subjective element to this type of study, but appreciate that in psychology there is little alternative.

This relatively small study of 36 post-menopausal women comprised 81% white subjects. Given the recent observations that ethnicity impacts on telomere length, it would have been sensible to include subjects from a single ethnic group to reduce the number of confounding variables. (There is no mention of any impact of ethnicity on telomere length, or if it was even considered as a confounder.)

There is also evidence that telomere attrition rates may differ in pre- vs. post-menopausal woman, a phenomenon that may be related to telomerase stimulation by estrogen. Therefore, it is pleasing to see that all subjects were post-menopausal (the authors have previously correlated perceived stress in post-menopausal women with shorter telomeres, but stress that this study is a different cohort).

The study reported a significant, negative association between IL-6 and telomere length, although the authors clearly state that this is not proof of causality: it may be that it is the critical shortening of telomeres that triggers inflammation rather that the other way around. Of the psychological measures optimism was not associated with either telomere length or IL-6, while pessimism is associated with both. This observation feels slightly counter-intuitive; is reduced pessimism not the same as increased optimism? If so, one would expect that if pessimism associates with shorter telomeres then surely optimism would associate with longer telomeres? Clearly this is not the case. As in the previous Epel/Blackburn study, higher levels of perceived stress were also associated with shorter telomeres, leading to the conclusion that “exposure to psychological stressors in pessimists could contribute to to chronic low-level increases in circulating pro-inflammatory cytokines…contributing to telomere shortening”. While this was a small study, it does add weight to the hypothesis that an individual’s psychological disposition is a contributing factor to poor health outcomes.

Cause for further concern for our downbeat friends is that an individual’s score on optimism and pessimism measures generally remain stable across time (important to note that this was not assessed in the present study, but has been replicated in earlier studies). Unfortunately for the happy folk out there, optimism again fails to associate with “better” health outcomes — so we may as well all take the middle-ground on this one.

Expression of pathogenic polyQ-repeat-containing huntingtin cripples a major endoplasmic reticulum quality assurance and garbage disposal pathway, ER-associated protein degradation (ERAD). The mutant protein appears to sequester essential components of the ERAD pathway. From Duennwald & Lindquist:

Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity

Protein misfolding, whether caused by aging, environmental factors, or genetic mutations, is a common basis for neurodegenerative diseases. The misfolding of proteins with abnormally long polyglutamine (polyQ) expansions causes several neurodegenerative disorders, such as Huntington’s disease (HD). Although many cellular pathways have been documented to be impaired in HD, the primary triggers of polyQ toxicity remain elusive. We report that yeast cells and neuron-like PC12 cells expressing polyQ-expanded huntingtin (htt) fragments display a surprisingly specific, immediate, and drastic defect in endoplasmic reticulum (ER)-associated degradation (ERAD). We further decipher the mechanistic basis for this defect in ERAD: the entrapment of the essential ERAD proteins Npl4, Ufd1, and p97 by polyQ-expanded htt fragments. In both yeast and mammalian neuron-like cells, overexpression of Npl4 and Ufd1 ameliorates polyQ toxicity. Our results establish that impaired ER protein homeostasis is a broad and highly conserved contributor to polyQ toxicity in yeast, in PC12 cells, and, importantly, in striatal cells expressing full-length polyQ-expanded huntingtin.

Long-lived organisms tend to be resistant to many types of stress, whereas short-lived organisms tend to be stress sensitive. This happy coincidence allows us to screen for longevity mutants by looking for stress resistance rather than long life (advantage: it takes a lot less time to do the primary screen).

The same logic ought to apply to small-molecule drugs: Any compound that increases stress resistance has an improved change of extending lifespan. That hypothesis has been operationally tested by the Lithgow lab, who performed a small-scale screen of antioxidant compounds and looked for molecules that increased thermotolerance in the worm C. elegans. Several of these drugs also increased lifespan. From Benedetti et al.:

Compounds that confer thermal stress resistance and extended lifespan

The observation that long-lived and relatively healthy animals can be obtained by simple genetic manipulation prompts the search for chemical compounds that have similar effects. Since aging is the most important risk factor for many socially and economically important diseases, the discovery of a wide range of chemical modulators of aging in model organisms could prompt new strategies for attacking age-related disease such as diabetes, cancer and neurodegenerative disorders … . Resistance to multiple types of stress is a common trait in long-lived genetic variants of a number of species; therefore, we have tested compounds that act as stress response mimetics. We have focused on compounds with antioxidant properties and identified those that confer thermal stress resistance in the nematode Caenorhabditis elegans. Some of these compounds (lipoic acid, propyl gallate, trolox and taxifolin) also extend the normal lifespan of this simple invertebrate, consistent with the general model that enhanced stress resistance slows aging.

Note that the authors tested resistance to thermal, rather than oxidative stress — given their choice to screen only antioxidant compounds, to do the latter would have been a bit circular. Still, given the history of antioxidant compounds as candidate anti-aging compounds, and the widespread belief that reactive oxygen species per se are a causative force in aging, the decision to screen only antioxidants does raise the possibility that the lifespan extension is due to the antioxidant activity of these compounds and that the stress resistance is merely an epiphenomenon.

Then again, it’s quite impressive that so many different antioxidants of so many different types can confer thermotolerance and increased longevity, and suggests that perhaps the association between antioxidants and longevity may have never had much to do with oxidation as such, but rather with some as-yet-uncovered connection between antioxidants and the activation of stress response pathways.

This is not, sensu stricto, a post about aging, but what can I say? Tardigrades! I love these little guys. Plus it’s Friday.

Last year we learned that organisms of the phylum Tardigrada (the so-called “water bears,” most closely related to arthropods) are unusually resistant to physiological stress. Given the well-established relationship between long lifespans and resistance to (most) stresses, I had wondered why tardigrades are not also unusually long-lived. (They can persist for years in a dormant state, but their fully animated lifespans are on the order of months.)

Now, Jönsson et al. reveals that tardigrades can survive unshielded in outer space:

Tardigrades survive exposure to space in low Earth orbit

Vacuum (imposing extreme dehydration) and solar/galactic cosmic radiation prevent survival of most organisms in space. Only anhydrobiotic organisms, which have evolved adaptations to survive more or less complete desiccation, have a potential to survive space vacuum, and few organisms can stand the unfiltered solar radiation in space. Tardigrades, commonly known as water-bears, are among the most desiccation and radiation-tolerant animals and have been shown to survive extreme levels of ionizing radiation. Here, we show that tardigrades are also able to survive space vacuum without loss in survival, and that some specimens even recovered after combined exposure to space vacuum and solar radiation. These results add the first animal to the exclusive and short list of organisms that have survived such exposure.

Which is pretty cool, when you think about it.

Still, as an adherent of the “stress resistance = longevity” school, I am nagged by the question: If tardigrades can survive hard vacuum, ultra-low temperatures, blazing radiation and Klingon disruptors, what on Earth (or off it, for that matter) is going on inside their cells that does them in after a few short months of life?

Attentive readers will notice that I’ve skipped a couple of sessions: Session III was “oral presentations from abstracts,” a series of unrelated short talks; Session IV was a poster session, and Session V was a series of talks about mitochondria that I watched from the bar, where taking out my laptop might have risked a catastrophic beer spill, so I didn’t blog it.

This morning is a session on subjects near and dear to my own heart.

John Sedivy kicked off the series with a discussion of assays for detecting cellular senescence in vivo and in vitro. This is an incredibly important subject, since the current assays have a lot of problems, ranging from poor quantifiability to frank irreproducibility. The Sedivy lab has developed a large number of image-processing protocols that will allow reliable detection and quantitation in multiple system. These techniques will hopefully allow us to nail down once and for all the location, origins, fate, and function of senescent cells in aging tissues. (Heidi Scrable said during questions that her lab has developed a way to do immunohistochemistry after SA ß-gal, a twitchy technique that usually ruins a sample for subsequent analysis; I will definitely be going to that poster this afternoon.)

Next up was Darren Baker, who is doing genetics with progeroid mice: In the BubR1 progeria model, loss of p19 or p53 results in accelerated aging, implying that p53 is involved in delaying aging in vivo. This directly contradicts the idea that p53-induced senescence is a cause of aging, and enters the fray on the side of scholars who believe that properly regulated p53 has a primarily anti-aging function.

Alex Bazarov asked whether p16-induced senescence is reversible in breast cancer cells (it isn’t), and proposed using a small molecule inducer of p16 arrest as a cancer therapy.

Oliver Bischof demonstrated that repetitive DNA is transcribed at the onset of senescence, generating a population of small noncoding RNAs that are sufficient to induce both senescence-associated heterochromatic foci (SAHFs) as well as the senescence growth arrest itself. Note that these small RNAs are distinct from micro-RNAs, whose role in senescence and cancer was the subject of several posters at this meeting (including one by me).

Kan Cao studies progerin, the derivative of Lamin A that is responsible for Hutchinson-Gilford Progeria Syndrome (HGPS). Today’s talk focuses on the role of progerin in normal aging: Normal cells express progressively more progerin as a function of age, but telomerase-immortalized cells express hardly any. Thus, there may be a synergy between telomere shortening and progerin induction during cellular senescence.

Norm Sharpless shared human genetic evidence that variations in the p16/INK4a locus are associated with variations in the rate of aging, cancer, and other age-related diseases (specifically atherosclerosis). The overall results suggest that p16 has diverse effects in different tissues.

Vera Gorbunova discussed the distinct tumor suppressor mechanisms that have evolved in rodents of varying body size and lifespans. She began by introducing the negative correlation between telomerase activity and body size, and between in vitro replicative lifespan and body size. Larger body size means more cell divisions and a greater cancer risk; hence replicative senescence is more common among larger rodents. Another sort of control is observed in naked mole rats, which are long-lived and whose cells exhibit multiple forms of contact-mediated growth arrest. I especially enjoyed the talk because of my recently stoked interest in comparative biogerontology.

On to other exotic organisms: Fish! Our finned friends are getting into the biogerontological act — it was only a matter of time. Shuji Kishi talked about genetic screens in zebrafish that identified mutants showing alterations in senescence-associated biomarkers (specifically, the senescence-associated beta-galactosidase, aka SA ß-gal). One of the mutants he described is deficient in telomere maintenance, and exhibits segmental progeria as well as shortened lifespan; another mutant causes accumulation of lipofuscins, suggesting a defect in lysosomal metabolism or autophagy.

Valery Krizhanovsky, who works right here at Cold Spring Harbor in Scott Lowe’s lab, described a useful function for cellular senescence beyond its well-documented tumor-suppressor function: prevention of liver fibrosis. Senescent cells are present in fibrotic liver in wildtype animals, but in cell-specific p53 knockouts these senescent cells are missing. The senescent hepatic cells appear to attract immune infiltration, which work to clear the senescent cells and in the process alleviate the fibrosis.

Francis Rodier (from the Campisi lab, where I also work) presented his work on the relationship between persistent DNA damage, senescence growth arrest, and the senescence-associated secretory phenotype (SASP). He focused on the regulation of the SASP by an upstream kinase in the DNA damage response pathway — a seminal example of the connection between the chromatin lesions in a compromised genome and the regulation of cell-cell communication.


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