(previous session)

Craig Skinner (Lin Lab, UC Davis): Identification of potential calorie restriction mimics in yeast using a nitric oxide-based screen. Yeast are an important model system in biogerontology, useful not only for genetic studies of longevity control but also for discovery of bioactive compounds. Calorie restriction (CR) in yeast causes increased levels of nitric oxide (NO) — somewhat surprising in that yeast cells lack a homolog of nitric oxide synthase — and elevated NO is sufficient to extend yeast lifespan. These observations led Skinner to screen a yeast deletion library for elevated NO levels, yielding several genes that extend lifespan.

Mark Lucanic (Lithgow Lab, Buck): Endocannabinoid signaling mediates the effect of diet on lifespan in C. elegans. Mutants in the dauer pathway in C. elegans often influence longevity; the daf-2 mutation, which causes constitutive dauer formation at elevated temperatures, extends lifespan by several fold. Lucanic discovered that endocannabinoids are involved in the regulation of the dauer pathway — and therefore, of longevity — either independently of or far downstream of daf-2 and daf-16. Endocannabinoids are upregulated under well-fed conditions, and shorten lifespan.

Delia David (Kenyon Lab, UCSF): Widespread protein aggregation is an inherent part of aging in C. elegans. Protein aggregates are a hallmark of many age-related neurodegenerative diseases, leading to the hypotheses that the cellular mileu changes with age in a manner that causes native, aggregation-prone proteins to form aggregates. David used mass spectrometry to identify a subset of normal worm proteins aggregate as a function of age. As with the proteins associated with neurodegeneration, specific proteins aggregate in specific cell types. Mutations that extend lifespan (such as daf-2) decrease aggregation, and tend to downregulate the expression of genes encoding aggregation-prone proteins. Curiously, regulators of protein homeostasis tend to aggregate themselves, leading to a destructive positive feedback loop in which the very factors that protect the cell from proteotoxicity disappear into aggregates, leading to further aggregation.

Cherry Tang (Zhong Lab, Berkeley): The Clearance of Ubiquitinated Protein Aggregates Via Autophagy. Autophagic protein degradation has been implicated in control of lifespan: autophagy slows cell and tissue aging. Tang has identified a protein that participates in degradation of ubiquitinated proteins and co-localizes with autophagosomes; when the protein is knocked down, protein aggregates become more toxic.

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As I was wandering the net today I found a very nice writeup about the 2009 report of an association between the FOXO3A gene and human aging. I found the article at the apparently quite popular but new-to-me blog Singularity Hub.

We mentioned this work in a brief post last year. The overall conclusion is that natural variants in this gene that are associated with extreme longevity. (The FOXO3A gene is a homolog of DAF-16, a longevity determinant in worms.) The 2009 paper describes a study of German centenarians, and is consistent with similar results in Japanese-Americans, published in 2008. Other genetic variants associated with lifespan include the hTERT and hTERC loci, recently described in a study of Ashkenazi Jewish centenarians.

Mostly I’m writing this post to introduce our readers to an interesting site: Singularity Hub contains a lot of excellent biogerontology coverage (in their longevity category). Much of the writing on that topic is by senior editor Aaron Saenz, who does a great job of critically addressing the newest findings in a very reader-friendly and accessible style. I’m going to subscribe to their feed and start reading regularly. Overall it’s a very professional and well-written site, and I’d recommend it to Ouroboros readers.

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Not only does the mammalian sirtuin SIRT1 mediate the lifespan extension phenotype of caloric restriction (CR), it is also involved in controlling behavior (such as food intake) in response to CR (and possibly during ad libitum feeding as well).

Two recent papers with consistent results address the issue. Both studies employed brain-specific knockouts of SIRT1; Cohen et al. used a brain-specific knockout, whereas Çakir et al. used both pharmacologic inhibition and an siRNA in the hypothalamus. The latter paper implicates the FoxO1 transcription factor and S6 kinase signaling, implying cross-talk with both the IGF-1 and TOR pathways.

ResearchBlogging.orgÇakir, I., Perello, M., Lansari, O., Messier, N., Vaslet, C., & Nillni, E. (2009). Hypothalamic Sirt1 Regulates Food Intake in a Rodent Model System PLoS ONE, 4 (12) DOI: 10.1371/journal.pone.0008322

Cohen, D., Supinski, A., Bonkowski, M., Donmez, G., & Guarente, L. (2009). Neuronal SIRT1 regulates endocrine and behavioral responses to calorie restriction Genes & Development, 23 (24), 2812-2817 DOI: 10.1101/gad.1839209

We are all descendents of an unbroken line of cell divisions, dating back to the last common ancestor of all life on Earth. At some point, long after our lineage had acquired features like nuclei and mitochondria, a less distant ancestor stumbled on a major innovation: it grew a body, bringing with it the advantages of cell and tissue specialization.

For many multicellular organisms, this specialization included a distinction between the mortal cells (the “soma”) and the potentially immortal cells (the “germ line”) that are capable of participating in the creation of new organisms. When you look at us, most of what you see is soma — the germ line is safely tucked away in the gonad, which is (usually) itself tucked away someplace safe.

But both the germ line and soma are made of cells. How is it that the soma is mortal while the germ line is, for practical purposes, immortal?

The disposable soma theory of aging begins from the premise that an organism has access to a finite amount of resources (broadly, energy and matter), and that it must distribute these resources in a way that maximizes reproductive fitness. First dibs goes to the germ line (without which it doesn’t matter, in a fitness sense, what becomes of the rest of the organism) and the rest gets divided among the cells of the soma.

For the moment, all we really need to take away from this model is that the germ line and soma are maintained in different ways, either in quality or extent. The germ line is doing something differently than the soma, the upshot of which is that the germ line is immortal. (A strict interpreter of the theory would presume that this “something” is resource-intensive, so that it wouldn’t be possible to apply the strategy to the soma. It’s also possible, however, that it’s simply inconsistent with optimal somatic functions — e.g., that making a muscle the best muscle it can be requires that myocytes not partake of the germ line strategy for immortality, for some structural reason that has nothing to do with resource allocation per se.)

One oh-wow corollary of this model is that if somatic cells could be made more like germ line cells, they would live longer. This prediction has a deliciously outrageous quality — yet is so simple that upon first hearing it, I reached for the nearest journal with the intention of rolling it up and smacking myself repeatedly on the forehead. Fortunately, there was a copy of Nature handy.

To be honest, it didn’t really happen that way. That copy of Nature contained the very article that introduced me to this concept: Curran et al. have shown that in long-lived mutants of the worm C. elegans, somatic tissues start acting like germ line cells:

A soma-to-germline transformation in long-lived Caenorhabditis elegans mutants

Unlike the soma, which ages during the lifespan of multicellular organisms, the germ line traces an essentially immortal lineage. Genomic instability in somatic cells increases with age, and this decline in somatic maintenance might be regulated to facilitate resource reallocation towards reproduction at the expense of cellular senescence. Here we show that Caenorhabditis elegans mutants with increased longevity exhibit a soma-to-germline transformation of gene expression programs normally limited to the germ line. Decreased insulin-like signalling causes the somatic misexpression of the germline-limited pie-1 and pgl family of genes in intestinal and ectodermal tissues. The forkhead boxO1A (FOXO) transcription factor DAF-16, the major transcriptional effector of insulin-like signalling, regulates pie-1 expression by directly binding to the pie-1 promoter. The somatic tissues of insulin-like mutants are more germline-like and protected from genotoxic stress. Gene inactivation of components of the cytosolic chaperonin complex that induce increased longevity also causes somatic misexpression of PGL-1. These results indicate that the acquisition of germline characteristics by the somatic cells of C. elegans mutants with increased longevity contributes to their increased health and survival.

Just to be clear: the somatic tissues of the long-lived mutants had not actually transformed into germ line cells as such, nor were the mutant worms festooned with extra gonads (though admittedly, that would be totally awesome). Rather, the somatic tissues exhibited gene expression patterns ordinarily found only in the germ line.

On the correlation vs. causation issue: The authors showed, using RNAi knockdowns, that the germ line-restricted genes were required for the longevity enhancement due to the mutation in daf-2 (worm insulin/IGF). There’s a bit of a wrinkle: in wildtype animals, blocking these same genes actually resulted in an increase in lifespan. How to explain that? The proffered rationale is that in the wildtype, germ line-restricted genes are only present in the germ line. Knocking them down has no effect on somatic tissue, but might reduce the activity of germ line cells; it’s been known for some time that ablating part of the gonad has life-extending consequences in wildtype animals.

The critical observation, in any case, is that the germ line genes are turned on in daf-2 mutants, and this activation is necessary in order for daf-2 mutation to extend lifespan.

Next questions, in rough order of difficulty:

  1. Does the soma-to-germ line transition occur in other long-lived mutants, or in calorie restricted animals?
  2. By what mechanisms are the germ line-restricted genes extending the somatic lifespan?
  3. Will this finding generalize to other metazoans?
  4. Do the germ line genes expressed in daf-2 soma contribute to germ line immortality?

ResearchBlogging.orgCurran, S., Wu, X., Riedel, C., & Ruvkun, G. (2009). A soma-to-germline transformation in long-lived Caenorhabditis elegans mutants Nature DOI: 10.1038/nature08106

Protein degradation is an essential longevity assurance pathway. Maintaining high levels of autophagy can delay age-related decline in liver function. Obstacles to protein degradation tend to shorten the lifespan: blocking autophagy causes hypersensitivity to stress, and inhibiting the ubiquitin/proteasome pathway damages the mitochondria; both of these treatments kill neurons.

Conversely, longevity enhancement tends to enhance disposal of cellular garbage: In a worm model of Alzheimer’s disease, long-lived daf-2 mutants exhibit slower protein aggregation and decreased proteotoxicity, probably as a result of higher rates of protein degradation.

Despite the overall importance of protein degradation in delaying aging, the destruction of individual proteins is not always a good thing. During a screen of worm E3 ubiquitin ligases, Mehta et al. discovered that blocking the degradation of the HIF-1 protein dramatically increases lifespan and blocked the toxicity of pathogenic, aggregation-prone proteins.

Proteasomal Regulation of the Hypoxic Response Modulates Aging in C. elegans

The Caenorhabditis elegans von Hippel-Lindau tumor suppressor homolog VHL-1 is a cullin E3 ubiquitin ligase that negatively regulates the hypoxic response by promoting ubiquitination and degradation of the hypoxic response transcription factor HIF-1. Here, we report that loss of VHL-1 significantly increased life span and enhanced resistance to polyglutamine and amyloid beta toxicity. Deletion of HIF-1 was epistatic to VHL-1, indicating that HIF-1 acts downstream of VHL-1 to modulate aging and proteotoxicity. VHL-1 and HIF-1 control longevity by a mechanism distinct from both dietary restriction and insulin/IGF-1-like signaling. These findings define VHL-1 and the hypoxic response as an alternative longevity and protein homeostasis pathway.

The initial finding was that knockdowns of the E3 ligase VHL-1 were long-lived. VHL-1 is known to degrade HIF-1, the transcription factor involved in the hypoxic response. To rule out the possibility that another substrate of VHL-1 was important in the longevity enhancement, the authors used fairly straightforward genetic analysis: Mutating EGL-9, another gene required for HIF-1 degradation, also confers the lifespan extension, but neither vhl-1 not egl- mutants could live long in the absence of HIF-1.

The VHL-1/EGL-9/HIF-1 pathway is distinct from other means of lifespan extension: both daf-2 mutants and calorie restricted animals could extend the lifespan of hif-1 mutants, and conversely vhl-1 mutations could further extend the lifespan of daf-2 animals. This distinction may exist only at the level of the more upstream signaling events, however: DAF-16, the longevity assurance transcription factor that is disinhibited by daf-2 mutation, shares many target genes with HIF-1, so it is possible that the longevity enhancements rely on the same stable of stress-resistance and repair genes.

Will boosting HIF-1 levels also influence lifespan in mammals? Probably not, at least not in any simple way: the proteins involved are highly conserved — but VHL-1 is a tumor suppressor in humans, so targeting it with a drug is almost definitely a bad idea. Since suppression of the hypoxic response (especially angiogenesis) is likely to be important to the mechanism of tumor suppression by VHL-1, the same goes for HIF-1. It wouldn’t be incredibly surprising if this particular mechanism of lifespan regulation weren’t conserved between worms and mammals: worms don’t like oxygen as much as we do, so even if the machinery is conserved, the physiological consequences of activating that machinery might not be.

Still, as the authors point out, there might be some value in exploring manipulations of the hypoxic response in post-mitotic tissue – like brain — where the risk of tumorigenesis would presumably be smaller.

ResearchBlogging.orgMehta, R., Steinkraus, K., Sutphin, G., Ramos, F., Shamieh, L., Huh, A., Davis, C., Chandler-Brown, D., & Kaeberlein, M. (2009). Proteasomal Regulation of the Hypoxic Response Modulates Aging in C. elegans Science DOI: 10.1126/science.1173507

The insulin-like growth factor (IGF) pathway is one of the longest-known and well-studied regulators of longevity. Extracellular signals (insulin-like peptides) activate insulin-receptor homologs (in worm, DAF-2) which in turn recruit and activate phosphoinositol 3-kinases (AGE-1). PI3Ks convert PIP2 into PIP3, which tethers and recruits other kinases such as AKT-1. Eventually, activation of these upstream kinases results in phosphorylation and inactivation of the longevity assurance gene DAF-16, which encodes a transcription factor that activates (among many other things) stress resistance genes.

To recap: High DAF-2 and AGE-1 activity => low DAF-16 activity => shorter lifespan. Lower DAF-2 or AGE-1 => high DAF-16 activity => longer lifespan. The lifespan extension of daf-2 or age-1 mutants absolutely requires wildtype DAF-16.

From this simple model, it would seem that the levels of DAF-2 agonists would run the show; DAF-16 activity would simply be a readout of signaling upstream of the insulin-like growth factor receptor. As is so often the case, however, this simple model turns out to be simplistic: DAF-16 plays an active role in determining the signaling through this pathway, as revealed by Tazearslan et al.:

Positive Feedback between Transcriptional and Kinase Suppression in Nematodes with Extraordinary Longevity and Stress Resistance

Insulin/IGF-1 signaling (IIS) regulates development and metabolism, and modulates aging, of Caenorhabditis elegans. In nematodes, as in mammals, IIS is understood to operate through a kinase-phosphorylation cascade that inactivates the DAF-16/FOXO transcription factor. Situated at the center of this pathway, phosphatidylinositol 3-kinase (PI3K) phosphorylates PIP2 to form PIP3, a phospholipid required for membrane tethering and activation of many signaling molecules. Nonsense mutants of age-1, the nematode gene encoding the class-I catalytic subunit of PI3K, produce only a truncated protein lacking the kinase domain, and yet confer 10-fold greater longevity on second-generation (F2) homozygotes, and comparable gains in stress resistance. Their F1 parents, like weaker age-1 mutants, are far less robust—implying that maternally contributed trace amounts of PI3K activity or of PIP3 block the extreme age-1 phenotypes. We find that F2-mutant adults have <10% of wild-type kinase activity in vitro and <60% of normal phosphoprotein levels in vivo. Inactivation of PI3K not only disrupts PIP3-dependent kinase signaling, but surprisingly also attenuates transcripts of numerous IIS components, even upstream of PI3K, and those of signaling molecules that cross-talk with IIS. The age-1(mg44) nonsense mutation results, in F2 adults, in changes to kinase profiles and to expression levels of multiple transcripts that distinguish this mutant from F1 age-1 homozygotes, a weaker age-1 mutant, or wild-type adults. Most but not all of those changes are reversed by a second mutation to daf-16, implicating both DAF-16/ FOXO–dependent and –independent mechanisms. RNAi, silencing genes that are downregulated in long-lived worms, improves oxidative-stress resistance of wild-type adults. It is therefore plausible that attenuation of those genes in age-1(mg44)-F2 adults contributes to their exceptional survival. IIS in nematodes (and presumably in other species) thus involves transcriptional as well as kinase regulation in a positive-feedback circuit, favoring either survival or reproduction. Hyperlongevity of strong age-1(mg44) mutants may result from their inability to reset this molecular switch to the reproductive mode.

PIP3 is necessary for the membrane tethering and activation of a great many kinases; thus, profound defects in PI3K activity would be predicted to result in profound defects in phosphoprotein signaling. Indeed, the authors see dramatic effects on in vitro kinase activity and steady-state phosphoprotein levels in their age-1 mutants — not just for known downstream targets of the IGF pathway but for bulk protein.

Note the importance of using F2 age-1 mutants, i.e., animals that are themselves the offspring of homozygous age-1 mutants: Wildtype AGE-1 activity in the parent, even from a single copy of the gene, is sufficient to maternally rescue PI3K activity in the offspring to some extent. This results in a weaker phenotype in the F1s than in their progeny, the F2s, who completely lack PI3K activity and consequently enjoy much longer lifespan and higher resistance to stress. There’s no way to find out about F3s, since the F2s are completely sterile.

In the absence of upstream IGF signaling, downstream effector kinases would not be activated by phosphorylation. Here’s where the story throws us a curve-ball: As predicted, in the profoundly long-lived age-1 F2’s, the effector kinases are inactive — but the transcripts encoding them are also downregulated. In the absence of upstream signaling, there’s no longer a kinase cascade bearing down on DAF-16, which therefore remains unphosphorylated and active. And what does DAF-16 do? It heads to the nucleus and transcriptionally silences the genes encoding the upstream kinases DAF-2, AGE-1 and others — in other words, DAF-16 turns off the genes that could turn off DAF-16.

It’s a feedback loop! Disinhibition of DAF-16 by lowering PIP3 levels is self-sustaining, because disinhibited DAF-16 lowers transcription of PI3Ks, thereby further lowering PIP3 levels. The authors argue that this arrangement represents a biological switch between a short-lived “reproductive state” and a non-reproducing “longevity state”, characterized by DAF-16 activation of stress-resistance and other types of longevity assurance genes.

They have a point, but I think they might be overstating the “switchiness” of this switch. One of the main features of a switch (of the sort that motivates the analogy) is that it can be on or off but not halfway between the two — there are disequilibrating forces at work that push it away from the middle and toward either pole. Unfortunately for the authors’ interpretation, most of the work done in this field to date has been done in the equivalent of the F1 age-1 mutants, where a combination of partial gene function and maternal factors have placed worms somewhere between “all reproduction” and “all longevity”. The existence of these animals does somewhat mitigate the argument that this system represents a binary switch in the strictest sense of the word.

To be fair to the authors, they acknowledge this, most clearly in Figure 6, a model that allows for three states — reproductive (low DAF-16, e.g., wildtype), longevity (high DAF-16, e.g. daf-2 or age-1 F1), and “hyperlongevity” (extremely high DAF-16 that completely shuts off PI3K activity, e.g. age-1 F2). The distinction between the two long-lived cases is somewhat elided in the Discussion, where the authors emphasize the feedback loop and consider the longevity states as though they were the same.

ResearchBlogging.orgTazearslan, C., Ayyadevara, S., Bharill, P., & Shmookler Reis, R. (2009). Positive Feedback between Transcriptional and Kinase Suppression in Nematodes with Extraordinary Longevity and Stress Resistance PLoS Genetics, 5 (4) DOI: 10.1371/journal.pgen.1000452

The CLK-1 gene is a longevity regulator that has been conserved across evolution: loss of function in both C. elegans (where the gene was first described) and hemizygosity in mouse (where the gene is called mCLK1) results inincreased lifespan (though at a cost to evolutionary fitness). Consistent with this, at least one anti-neurodegeneration drug inhibits mCLK1, raising the possibility that the drug acts by delaying aging and thereby postponing age-related neurological disease. Genetic crosses and other evidence suggest that the longevity extension is independent of the action of the insulin-like growth factor (IGF)/DAF-2 pathway, another conserved regulator of longevity.

The CLK-1 protein catalyzes a late step in the biosynthesis of ubiquinone (aka coenzyme Q or coQ), an essential cofactor in mitochondrial electron transport, but it’s not completely clear whether this enzymatic function has to do with the life-extension phenotype. In worm, even among clk-1 mutants that completely lack detectable coQ and accumulate the same levels of the metabolic precursor DMQ, null mutants (which make no CLK-1 protein) exhibit more severe phenotypes than missense mutants (which express normal levels of a defective protein). Furthermore, tRNA suppressor mutations appear to differentially affect the lifespan, developmental and ubiquinone synthesis phenotypes. Finally, dietary supplementation with coQ fails to rescue the developmental and lifespan phenotypes (though this is confounded by the fact that these worms never accumulate cellular levels of coQ that are comparable to wildtype). (For a more detailed treatment of CLK-1 biology in both worm and mouse, see this excellent review by Stepanyan et al.).

How, then, do clk-1 mutants extend longevity? To address this question, Cristina et al. compared the the gene expression profiles of wildtype and clk-1 worms:

A regulated response to impaired respiration slows behavioral rates and increases lifespan in Caenorhabditis elegans

When mitochondrial respiration or ubiquinone production is inhibited in Caenorhabditis elegans, behavioral rates are slowed and lifespan is extended. Here, we show that these perturbations increase the expression of cell-protective and metabolic genes and the abundance of mitochondrial DNA. This response is similar to the response triggered by inhibiting respiration in yeast and mammalian cells, termed the “retrograde response”. As in yeast, genes switched on in C. elegans mitochondrial mutants extend lifespan, suggesting an underlying evolutionary conservation of mechanism. Inhibition of fstr-1, a potential signaling gene that is up-regulated in clk-1 (ubiquinone-defective) mutants, and its close homolog fstr-2 prevents the expression of many retrograde-response genes and accelerates clk-1 behavioral and aging rates. Thus, clk-1 mutants live in “slow motion” because of a fstr-1/2-dependent pathway that responds to ubiquinone. Loss of fstr-1/2 does not suppress the phenotypes of all long-lived mitochondrial mutants. Thus, although different mitochondrial perturbations activate similar transcriptional and physiological responses, they do so in different ways.

The transcriptional phenotype of clk-1 mutant animals is similar to the phenotype observed when mitochondrial respiration is inhibited; this is the “retrograde response” mentioned in the abstract (so called because the signal travels from the mitochondria to the nucleus rather than in the other direction). Importantly, blocking the retrograde response by knocking down two effector molecules (FSTR-1 and -2) both prevents many of these transcriptional changes and rescues the lifespan phenotype — strongly implying that the retrograde response is inducing genes that contribute to the enhanced longevity of the mutants.

Taken together, I think these findings make a strong argument that coQ synthesis and lifespan phenotypes are closely related. The observation that clk-1 mutants phenocopy inhibition of respiration is not surprising when one recalls that coQ is a key mitochondrial cofactor. If knocking down respiration causes life-extending transcriptional changes, and reduction in coQ levels knocks down respiration, then it follows that the reduction in coQ levels is the ultimate cause of lifespan extension in clk-1 mutants.

There is a danger that we’re seeing the forest but missing a specific tree: The transcriptional responses to clk-1 mutation and inhibited respiration are not identical. It remains a formal possibility that wildtype CLK-1 mediates lifespan effects via one of the genes that is regulated (in a FSTR-1/2-dependent manner) in clk-1 but not during the retrograde response, and that the similarity to the retrograde response is a red herring. This is a tough sell, however, since one must still account for the growing body of evidence (cited by Cristina et al.) that inhibition of respiration by other means can also increase longevity.

ResearchBlogging.orgCristina, D., Cary, M., Lunceford, A., Clarke, C., & Kenyon, C. (2009). A Regulated Response to Impaired Respiration Slows Behavioral Rates and Increases Lifespan in Caenorhabditis elegans PLoS Genetics, 5 (4) DOI: 10.1371/journal.pgen.1000450

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