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This annual review focuses on invertebrate model organisms, which shed light on new mechanisms in aging and provide excellent systems for both genome-wide and in-depth analysis. This year, protein interaction networks have been used in a new bioinformatic approach to identify novel genes that extend replicative lifespan in yeast. In an extended approach, using a new, human protein interaction network, information from the invertebrates was used to identify new, candidate genes for lifespan extension and their orthologues were validated in the nematode Caenorhabditis elegans. Chemosensation of diffusible substances from bacteria has been shown to limit lifespan in C. elegans, while a systematic study of the different methods used to implement dietary restriction in the worm has shown that they involve mechanisms that are partially distinct and partially overlapping, providing important clarification for addressing whether or not they are conserved in other organisms. A new theoretical model for the evolution of rejuvenating cell division has shown that asymmetrical division for either cell size or for damaged cell constituents results in increased fitness for most realistic levels of cellular protein damage. Work on aging-related disease has both refined our understanding of the mechanisms underlying one route to the development of Parkinson’s disease and has revealed that in worms, as in mice, dietary restriction is protective against cellular proteotoxicity. Two systematic studies genetically manipulating the superoxide dismutases of C. elegans support the idea that damage from superoxide plays little or no role in aging in this organism, and have prompted discussion of other kinds of damage and other kinds of mechanisms for producing aging-related decline in function.
Invertebrate model organisms are powerful tools for aging research because of their relatively short lifespans, powerful ‘omic’ databases and ease of genetic analysis. This year, a novel bioinformatic approach has been used to identify candidate genes for increasing replicative lifespan (RLS) in yeast (Managbanag et al., 2008). The protein products of the set of 40 genes already shown, by either gain or loss of function, to increase RLS (longevity-assurance genes, LAGs) were mapped to a network of validated protein–protein interactions. In a shortest-path approach, candidate genes connecting lifespan-extending genes to each other in the network were identified. Measurement of RLS in the yeast gene deletion strains available for this set of candidate genes showed them to be highly enriched for increased RLS (15.9% of strains as opposed to 4.3% in a random set). The original set of mutant LAGs that increased RLS were a mixture of gain and loss of function, and the set of candidate gene deletion strains was also highly enriched for shortened RLS (36% vs. 17% in a random set). The new genes identified by this approach included novel components of the TOR pathway and of the translation machinery, both already implicated in RLS in yeast, but also genes involved in other functions such as bud formation and mitochondrial genome maintenance (Managbanag et al., 2008).
This kind of bioinformatic approach could be particularly useful for organisms where genetic screens for lifespan are not feasible, such as humans. Indeed, Bell et al. (2009) compiled a list of genes that, when mutated, can increase lifespan in yeast, the nematode worm C. elegans, the fruit fly Drosophila melanogaster or the mouse. The protein products of the human orthologues of these LAGs in the model organisms were then mapped in a new protein-protein interaction network for humans. Both these human orthologues of the invertebrate LAGs and genes encoding proteins that interacted with them (human interacting proteins), were enriched in the set of genes that change in RNA transcript expression with age in human muscle. Furthermore, when the expression of a sample of the C. elegans orthologues of the human interacting proteins was knocked down by RNA interference, 33% of them extended the lifespan of the worm, a much higher proportion than would be expected by chance (Bell et al., 2009). There is thus a strong signature of evolutionary conservation of genetic effects on lifespan between the model organisms and humans, which will be of great value in identifying candidate genes for population genetic analysis in humans and for further investigation of function in a two-way interplay between humans and model organisms.
Dietary restriction (DR) extends lifespan in numerous organisms, and it ameliorates aging-related loss of function and pathology, both in invertebrates and rodents (Mair & Dillin, 2008). Great strides have been made in understanding the mechanisms by which DR extends lifespan in invertebrates, in part by fuller characterisation of different methods for implementing DR within each organism. Although diverse methods for reduction in food intake have been shown to extend lifespan and are referred to as DR, it is becoming clear that some of their effects on lifespan are mediated by mechanisms other than reduced food intake and that, even within a single organism, different methods of DR can involve different mechanisms. DR has been implemented in the C. elegans by using eat mutations, which have pharyngeal pumping defects, by reduction of the growth rate or density of the E. coli bacteria present on the surface of nutrient agar or in liquid culture, by axenic growth and by complete removal of the bacterial food source in early adulthood (Mair & Dillin, 2008; Mair et al., 2009). At least part of the extension of lifespan from removal of the bacterial food source does not require reduced food intake, because food-removal in later adulthood increases lifespan even though the control worms no longer feed at this age. A diffusible substance from the bacteria limits lifespan in the control worms, through a route other than the chemosensory neurons already demonstrated to influence longevity (Smith et al., 2008). A similar, purely sensory, limitation of lifespan by volatiles from yeast was previously discovered in Drosophila (Libert et al., 2007), and this evolutionary conservation prompts speculation that similar sensory mechanisms may be important in mediating the effects of DR in rodents.
Until recently, all methods of DR in the worm were thought to increase lifespan by a mechanism at least partially distinct from that of reduced insulin/IGF-like signalling (IIS), because the key IIS-responsive forkhead transcription factor effector, DAF-16, was not required for the responses to DR (Mair et al., 2009). However, a recently developed method of DR, in which the worms are transferred to fresh culture dishes of different concentrations of live bacteria every 2 days (sDR), does require daf-16 and the catalytic subunit of the AMP kinase (Greer et al., 2007). A systematic investigation of the genetic pathways by which different methods of DR extend lifespan in C. elegans (Greer & Brunet, 2009) showed that another method of DR, dilution of peptone on bacterial plates, which reduces bacterial growth, also requires daf-16 and the catalytic subunit of the AMP kinase. However, these genes are not required for lifespan extension by bacterial dilution in liquid medium or by mutation of eat. Conversely, the forkhead transcription factor FoxA/pha-4, the transcription factor skn-1 and the heat shock responsive transcription factor hsf-1, all shown to be required for the response to at least one other method of DR, are not required for the response to sDR. Interestingly, a functional clk-1 gene, which when mutated can increase lifespan and which functions in the biosynthetic pathway to ubiquinone, is required for the increase in lifespan in response to sDR and to eat mutants. These different methods of DR therefore increase lifespan by pathways that are partially distinct and partially overlapping. Possible reasons for the similarities and differences could involve different levels of intake of specific nutrients, different sensory pathways, different levels of pathogenicity or other forms of stress, and different times of initiation (Greer & Brunet, 2009). It is an open question if the mechanisms by which invertebrate lifespan responds to DR also play a role in extension of lifespan by DR in mammals. This kind of systematic investigation of the mechanisms at work in a single invertebrate is a valuable step towards elucidation of candidate mechanisms and hence addressing the important issue of evolutionary conservation.
Babies are born youthful, no matter what the age of their parents. Analysis of the possible selection pressures underlying the evolution of this purely phenotypic rejuvenation process throws light on the evolution of aging itself. The processes involved in rejuvenation in multicellular organisms have barely started to be investigated. Rejuvenation in bacteria and unicellular organisms has so far proved to be associated with asymmetrical cell division, either for size, or with one daughter cell inheriting more than half of the damaged cellular constituents from the parent, or both. A new theoretical model, based on segregation of damaged proteins, has thrown further light on how these asymmetries might have evolved (Erjavec et al., 2008). The model assumed that each new cell started life with an amount of total protein (its size) some of which was damaged and some intact. In this model new, intact protein could be formed by synthesis, intact protein could become damaged, and damaged protein could be degraded, with rate constants that could be varied in the model. Cells could divide only when they reached a certain size threshold, and the growth rate of the cell was depressed by the presence of increasing absolute amounts of damaged protein. These assumptions fit well with the known dynamics of cell growth and division in yeasts. The effects of asymmetrical division, either for total size or for the damaged protein, were considered using either senescence of the whole clone or population growth rate as measures of fitness: the results were qualitatively similar for the two fitness measures. The striking result was that asymmetrical division, either for size or for damaged protein, was beneficial at most realistic rates of protein damage, and resulted in the appearance of a lineage of larger or more damaged cells that aged and gave rise to rejuvenated smaller or less damaged offspring. Other models have shown that asymmetric division for damage can be beneficial under some circumstances, but the pervasiveness of this effect and the ubiquitous benefit of pure size asymmetry have not been seen previously. Interestingly, the same study showed that in fission yeast Saccharomyces pombe, which undergoes a cell division that appears superficially symmetrical, there is an unequal segregation of damaged proteins, associated with lower replicative lifespan in the more damaged offspring, just as the model predicted. Cases of pure asymmetrical division for size, on the other hand, have not so far been reported, although they may simply have not have been detected. In one of the few cases that has been thoroughly investigated, the asymmetrical division for size in budding yeast Saccharomyces cerevisiae, there is also asymmetrical segregation of damaged cell components (eg. Erjavec et al., 2007). It will be important to discover what, if any, role such damage-retention plays in other situations associated with phenotypic rejuvenation of offspring.
The invertebrates provide excellent systems for investigation of the mechanisms by which aging acts as the major risk factor for common, lethal diseases such as cancer, cardiovascular disease and neurodegeneration. One important contribution has been the development and analysis of invertebrate models of aging-related disease. Fly models have been particularly effective for analysis of the aetiology of Parkinson’s disease (PD), the commonest neurodegenerative disease in humans, and they may ultimately throw light on the reasons for the heterogeneity in the condition. PD is predominantly sporadic and aging-related. It is associated with loss of dopaminergic neurons and the presence in the remaining neurons of Lewy bodies, which contain α-synuclein, encoded by a gene associated with dominant, familial forms of the disease. Mutations in several other genes also lead to various familial forms of PD. Some, rarer familial and sporadic cases are caused by autosomal recessive mutations in two genes, encoding a kinase and an E3 ubiquitin ligase, and they are not associated with Lewy bodies. Studies in Drosophila have thrown considerable light on the normal function of these two genes. The Drosophila orthologues, pink1 and parkin, function in the same pathway to maintain mitochondrial integrity, in testes, muscle and dopaminergic neurons (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). A new study has elucidated how they do this (Deng et al., 2008). Mitochondrial fission and fusion in mammals are promoted by several GTPases that have Drosophila orthologues, and this study contributed new evidence that these GTPases function similarly in fly and mammals. Mutants in these fly orthologues also showed strong genetic interactions with pink1 and parkin, in both testes and the indirect flight muscles, strongly suggesting that pink1 and parkin act to promote fission or inhibit fusion of mitochondria. Exactly how they do this awaits investigation, but the results suggest that it should be determined if PD patients have abnormalities in mitochondrial dynamics.
DR in rodents not only extends lifespan, but also ameliorates most aspects of aging-related pathology and loss of function. Similar findings have been reported for long-lived mice mutant for the somatotropic axis (Bartke, 2009; Barzilai & Bartke, 2009; Ikeno et al., 2009) or for IIS (Selman et al., 2008). It thus seems that extension of lifespan by these interventions is associated with a broad-spectrum improvement in health during aging although, interestingly, DR in Drosophila may be an exception (Bhandari et al., 2007; Burger et al., 2007). An important hypothesis now being addressed in the invertebrate models organisms is that interventions that slow the aging process may protect against not only normal, aging-related decline but also the pathology associated with specific models of aging-related diseases. For instance, lifespan-extending mutations in the gene daf-2, which encodes the single worm insulin/IGF receptor, can ameliorate the pathology associated with worm models of both tumours (Pinkston et al., 2006; Pinkston-Gosse & Kenyon, 2007) and Alzheimer’s disease (Cohen et al., 2006). Now, it seems that DR in the worm can suppress the toxicity associated with three different models of neurodegenerative disease (Steinkraus et al., 2008). Several human neurodegenerative disorders, including Huntington’s disease, are caused by polyglutamine tract expansion. One model of these disorders in the worm expresses 35 consecutive glutamine residues fused to yellow fluorescent protein. A second model, of Alzheimer’s disease (AD), expresses the Aß42 peptide, implicated in AD in humans. A third provides a more general model of the effects of proteotoxicity by expressing an aggregation prone form of green fluorescent protein. All of these peptides are expressed in the musculature of the worm and lead to a progressive paralysis. Very strikingly, DR implemented through either bacterial deprivation or making the worms mutant for eat-2, rescued this paralysis, strongly supporting the idea that amelioration of the aging process can protect against more than one form of aging-related disease. DR has also been found to ameliorate pathology in mouse models of AD (Qin et al., 2006, 2008), and these parallel findings in C. elegans raises the possibility of making more rapid progress with analysis of mechanisms in the worm. Mutation of daf-2 also rescued paralysis in the AD model, as previously shown (Cohen et al., 2006), and in the model of polyglutamine toxicity. However, this rescue by mutation in daf-2, but not the rescue of paralysis in these models by DR, depended upon the presence of daf-16, which encodes the forkhead transcription factor effector of IIS in the worm. Reduced IIS and bacterial deprivation thus rescue the polyglutamine and AD pathology by distinct mechanisms. However, both the rescue of paralysis in these two models by bacterial deprivation and the rescue of paralysis in the AD model by mutations in daf-2 require the heat shock transcription factor hsf-1. Further molecular analysis of these models will reveal if the molecular mechanisms by which hsf-1 contributes to the rescue are the same in the two cases. These findings imply that DR in the worm may protect against diverse mechanisms of proteotoxicity in cells, and it will be important to extend these investigations both to other key tissues, such as the nervous system, and to test their generality with other models of disease in C. elegans and in other organisms.
The generally accepted view of the aging process is that it is caused by the accumulation of damage, to molecules, cells, tissues and the whole system. Ever since the oxygen free radical theory was first proposed (Harman, 1956) and subsequently refined to focus on the superoxide radical issuing from the mitochondrial electron transport chain (Harman, 1972), theoretical and empirical work has focussed on oxidative damage as a leading, candidate mechanism for causing aging. However, two recent studies with C. elegans (Doonan et al., 2008; Van Raamsdonk & Hekimi, 2009) have undermined any simple role for superoxide in limitation of worm lifespan. C. elegans has two isoforms of the cytosolic Cu/Zn superoxide dismutases (SODs) and two of the mitochondrial MnSODs, together with a single extracellular Cu/ZnSOD. This complexity has held up a full investigation of their role in the aging process, but now these isoforms have each been systematically deleted or knocked down by RNA interference and the effects on lifespan determined (Doonan et al., 2008; Van Raamsdonk & Hekimi, 2009). Deletion of the two MnSODs caused increased sensitivity to the superoxide generator paraquat and severe sensitivity to hyperoxia but either had no effect on lifespan, or even increased it, under normal conditions, a finding also reported in a third study (Honda et al., 2008). Nor were either of these MnSODs required for increase in lifespan in response to reduced IIS. These findings suggest that superoxide generated inside mitochondria does not contribute to aging-related damage in C. elegans. Deletion of each of the cytosolic Cu/Zn SODs either slightly reduced lifespan or had no effect, and elimination of one of the isoforms increased sensitivity to paraquat. Over-expression of one isoform, which led to a 2-fold increase in SOD activity, sometimes very slightly increased lifespan. Cytosolic SOD could therefore play a very minor role in the aging process, with SOD-1 protecting against the aging-related damage produced. Deletion or knock-down of the extracellular Cu/Zn SOD was without effect on lifespan. Even knock-down of three SODs simultaneously in various combinations did not reduce lifespan (Van Raamsdonk & Hekimi, 2009). These findings support the idea that superoxide generated in any of the three main cellular compartments is not limiting for lifespan in C. elegans, which contrasts with the situation in Drosophila and mice, where complete loss of at least one SOD isoform is severely deleterious or lethal (Muller et al., 2007). An obvious explanation of this difference, given the presence of multiple isoforms, could be compensatory up-regulation of other SODs in C. elegans. However, superoxide does not cross membranes, and so compensation cannot explain the findings that deletion of the single extracellular SOD or of both mitochondrial or of both cytosolic isoforms caused either no shortening (extracellular, mitochondrial) or at most a small shortening (cytosolic) of lifespan. Alternatively, other defence systems could protect against superoxide. However, the hypersensitivity of many of these mutants and mutant combinations to superoxide-generating chemicals and to hyperoxia, with no correlation between this hypersensitivity and lifespan, argues against this interpretation. Furthermore, knock-down of the predominant MnSOD increased oxidative damage to proteins, yet did not decrease lifespan. Clear experimental support for a role of superoxide in aging has not been forthcoming from worm, fly or mouse (Muller et al., 2007). Although there could be a variety of explanations for this, and unambiguous contrary evidence is also hard to obtain, the recent results from C. elegans do strongly suggest that damage from superoxide is not limiting for lifespan.
In a thoughtful review of the current status of the oxidative damage theory of aging in C. elegansGems & Doonan (2009) argue that the time is ripe to consider alternatives to the oxidative damage theory, and even the possibility that aging is not caused by damage at all. It has sometimes been suggested that we have reached ‘the end of the beginning’ in aging research. However, we may still have some way to go.