Age-1 Mutation Research Articles

PIP3-binding proteins promote age-dependent protein aggregation and limit survival in C. elegans.

Class-I phosphatidylinositol 3-kinase (PI3KI) converts phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 comprises two fatty-acid chains that embed in lipid-bilayer membranes, joined by glycerol to inositol triphosphate. Proteins with domains that specifically bind that head-group (e.g. pleckstrin-homology [PH] domains) are thus tethered to the inner plasma-membrane surface where they have an enhanced likelihood of interaction with other PIP3-bound proteins, in particular other components of their signaling pathways. Null alleles of the C. elegans age-1 gene, encoding the catalytic subunit of PI3KI, lack any detectable class-I PI3K activity and so cannot form PIP3. These mutant worms survive almost 10-fold longer than the longest-lived normal control, and are highly resistant to a variety of stresses including oxidative and electrophilic challenges. Traits associated with age-1 mutation are widely believed to be mediated through AKT-1, which requires PIP3 for both tethering and activation. Active AKT complex phosphorylates and thereby inactivates the DAF-16/FOXO transcription factor. However, extensive evidence indicates that pleiotropic effects of age-1-null mutations, including extreme longevity, cannot be explained by insulin like-receptor/AKT/FOXO signaling alone, suggesting involvement of other PIP3-binding proteins. We used ligand-affinity capture to identify membrane-bound proteins downstream of PI3KI that preferentially bind PIP3. Computer modeling supports a subset of candidate proteins predicted to directly bind PIP3 in preference to PIP2, and functional testing by RNAi knockdown confirmed candidates that partially mediate the stress-survival, aggregation-reducing and longevity benefits of PI3KI disruption. PIP3-specific candidate sets are highly enriched for proteins previously reported to affect translation, stress responses, lifespan, proteostasis, and lipid transport.

Genes required for the functions of olfactory AWA neuron regulate the longevity of Caenorhabditis elegans in an insulin/IGF signaling-dependent fashion

To investigate the interaction between the genes required for the functions of AWA olfactory neuron and insulin/IGF signaling in regulating the longevity of nematode Caenorhabditis elegans (C. elegans). The mutants that had loss-of-function mutation of the genes required for AWA, AWC, ASE, and AFD sensory neurons were employed. Lifespan, the speed of pharynx pumping, the intestinal autofluorescence, the dauer formation, and the brood size were examined. Rescue experiments were performed to confirm the role of the genes required for the functions of AWA neuron in regulating lifespan. Moreover, genetic interactions between genes required for the functions of AWA neuron and insulin/IGF signaling were investigated. Mutations of odr-7, odr-2, and odr-3 genes required for the functions of AWA neuron significantly increased the mean lifespan of nematodes and slowed the accumulation of intestinal autofluorescence. Besides, these mutations were closely associated with higher pumping rates during aging. However, mutation of odr-7, odr-2, or odr-3 did not obviously affect the brood size or the dauer formation, and the regulation of longevity by odr-7, odr-2, and odr-3 was temperature-independent. In contrast, mutations of genes required for the functions of ASE, AWC, and AFD sensory neurons did not influence the nematode lifespan. Moreover, expression of odr-7, odr-2 and odr-3 in AWA neuron could completely or largely restore the altered lifespan in odr-7, odr-2 and odr-3 mutants. Furthermore, genetic interaction assay demonstrated that the extended lifespan in odr-7 mutant could be suppressed by daf-16 mutation and enhanced by daf-2 or age-1 mutation, whereas mev-1 and pha-4 were not required for the long lifespan of odr-7 mutant. The genes required for the function of AWA sensory neuron could regulate the nematode longevity in an insulin/IGF signaling-dependent fashion in C. elegans.

Long-term starvation and ageing induce AGE-1/PI 3-kinase-dependent translocation of DAF-16/FOXO to the cytoplasm.

BackgroundThe provision of stress resistance diverts resources from development and reproduction and must therefore be tightly regulated. In Caenorhabditis elegans, the switch to increased stress resistance to promote survival through periods of starvation is regulated by the DAF-16/FOXO transcription factor. Reduction-of-function mutations in AGE-1, the C. elegans Class IA phosphoinositide 3-kinase (PI3K), increase lifespan and stress resistance in a daf-16 dependent manner. Class IA PI3Ks downregulate FOXOs by inducing their translocation to the cytoplasm. However, the circumstances under which AGE-1 is normally activated are unclear. To address this question we used C. elegans first stage larvae (L1s), which when starved enter a developmentally-arrested diapause stage until food is encountered.ResultsWe find that in L1s both starvation and daf-16 are necessary to confer resistance to oxidative stress in the form of hydrogen peroxide. Accordingly, DAF-16 is localised to cell nuclei after short-term starvation. However, after long-term starvation, DAF-16 unexpectedly translocates to the cytoplasm. This translocation requires functional age-1. H2O2 treatment can replicate the translocation and induce generation of the AGE-1 product PIP3. Because feeding reduces to zero in ageing adult C. elegans, these animals may also undergo long-term starvation. Consistent with our observation in L1s, DAF-16 also translocates to the cytoplasm in old adult worms in an age-1-dependent manner.ConclusionDAF-16 is activated in the starved L1 diapause. The translocation of DAF-16 to the cytoplasm after long-term starvation may be a feedback mechanism that prevents excessive expenditure on stress resistance. H2O2 is a candidate second messenger in this feedback mechanism. The lack of this response in age-1(hx546) mutants suggests a novel mechanism by which this mutation increases longevity.

SALTY WORMS?

Osmotic stress is a frequent experience, both for organisms and the cells that make them up. An organism's ability to osmoregulate successfully is key to its survival, and mechanisms of osmoregulation are intensively studied by some authors in this journal. A recent paper from Kevin Strange's group uses the tiny nematode worm to implicate a very unexpected gene in the ability to survive hypertonic stress, linking osmoregulation to the ageing process.Caenorhabditis elegans is a tiny soil-dwelling nematode that has become a favoured model organism for genetics. It might seem unlikely that useful physiology could be performed on something only a few tens of microns in diameter, but Strange nonetheless showed that C. elegans could readily adapt to hyper- or hypotonic environments. Previous work had shown that a class of C. elegans mutants that altered the organism's ability to form a long-lived, stress-tolerant larval stage, called the dauer larva, could greatly alter the animal's resistance to thermal, oxidative or hypoxic stress. Strange reasoned that this might also extend to osmotic stress and, sure enough, showed that one such mutant, daf-2, conferred greatly increased survival on agar plates containing up to 400 mmol l–1 NaCl. daf-2 encodes the worm's insulin receptor,so the implication is clear; disrupting insulin signalling actually enhances survival under stress.There are other players in the insulin signalling cascade, so to explore the genes responsible for stress tolerance in C. elegans, the authors tried these other genes singly and in combination. Mutations in age-1, a gene implicated in longevity that encodes PI3-kinase, showed a similar salt resistance phenotype to daf-2. However, mutations in daf-16, also identified in screens for genes that affect lifespan,could suppress these effects in double mutants carrying daf-2 and daf-16 or carrying age-1 and daf-16. That is,normal daf-16 is required for daf-2 or age-1mutants to be effective at conferring salt resistance. Over-expression of daf-16, which leads to increased longevity, also increases salt tolerance. So longevity, ageing and resistance to osmotic and other stressors are all interlinked.daf-16 encodes a FOXO transcription factor, which switches on other genes, and other workers had recently published a microarray list of genes upregulated by daf-16 over-expression. Some might be candidates for increasing survival after salt stress. Conspicuous in the list were the heat shock protein (hsp) genes, which protect organisms from stress-induced damage; by knocking these down with RNAi (particularly easy in C. elegans), several of the hsps were shown to be necessary for successful adaptation to high salt. Inspired by this result, the authors screened a further 222 target genes of daf-16 by RNAi and found that 10 of them were also important in salt response. Two of these were trehalose-6-phosphate synthases, which catalyse the formation of trehalose, an important osmolyte in many organisms.In summary, the authors propose that in normal worms, insulin signalling through the daf-2 insulin receptor activates the age-1 PI3 kinase, which in turn normally phosphorylates the daf-16/FOXO transcription factor to keep it out of the nucleus, stopping it from activating downstream genes. In mutants of insulin signalling, daf-16is able to enter the nucleus, allowing it to switch on a variety of genes that allow the animal to adapt to stress more easily. It is interesting to note that these genes are presently the darlings of the ageing research community,suggesting that ageing and osmoregulation may be linked. And as these genes are conserved right across to humans, there is the prospect that work on the tiny worm may have uncovered a general mechanism for response to hypertonic stress.

Characterization of a Life-Extending Mutation in age-2, a New Aging Gene in Caenorhabditis elegans

We have generated a life-extending mutation, yw23, in Caenorhabditis elegans. The mutation is in what appears to be a new aging gene, which we have designated age-2. When homozygous, yw23 produces an increase of mean and maximum life span of about 20% over that of the wild-type strain, N2. Strain HG23 [age-2(yw23)] was obtained by screening for longer life spans among 430 lines of nematodes two generations after exposure to the mutagen ethylmethanesulfonate. Strain HG231 [age-2(yw23)] was obtained after a single out-crossing of HG23 to N2. When compared with N2, HG231 exhibits normal motility, slightly higher swimming rates, reduced fertility (especially at higher temperatures), somewhat longer development times, and a slightly larger size at the time of first egg laying. A Gompertz analysis suggests that HG231 extends life span by reducing the initial mortality rate. In genetic crosses, yw23 complements other known aging mutants in C. elegans genes-age-1, daf-2, spe-26, clk-1, clk-2, clk-3, and gro-1. A double-mutant strain, HG284, combining mutations in age-1 and age-2, lives longer than animals with individual mutations in either age-1 or age-2, and exhibits a longer life span at 25 degrees C than at 20 degrees C.

Aging and resistance to oxidative damage in Caenorhabditis elegans.

The dauer larva state and the age-1 mutation, both of which extend life-span in Caenorhabditis elegans, were tested for hyperresistance to cellular damage that may be relevant to aging. The age-1 strain TJ401 displayed hyperresistance to oxidative stress relative to its parental strain. The activities of two enzymes that protect cells from oxidative damage, superoxide dismutase (SOD) and catalase, showed an age-dependent increase in mutant animals, which was not seen in the parental strain. These increases in activities paralleled the time course of the hyperresistance. The results are consistent with the age-1 gene product functioning as a negative regulator of SOD and catalase activities. In wild-type and age-1 dauer larvae, elevated levels of SOD activity, but not of catalase activity, were present when compared with young adults. The common increase in SOD activity prompted cloning the C. elegans Cu/Zn SOD gene. Its position on the physical map of the genome was in the region to which the age-1 gene has been genetically mapped, but it is unlikely that a mutation at the SOD locus confers the Age phenotype. Results support the free radical theory of aging by suggesting that the increased resistance to oxidative stress may be among the causes of increased longevity in both strain TJ401 and in the dauer larva.

Comparing mutants, selective breeding, and transgenics in the dissection of aging processes of Caenorhabditis elegans.

The genetic analysis of aging processes has matured in the last ten years with reports that long-lived strains of both fruit flies and nematodes have been developed. Several attempts to identify mutants in the fruit fly with increased longevity have failed and the reasons for these failures are analyzed. A major problem in obligate sexual species, such as the fruit fly, is the presence of inbreeding depression that makes the analysis of life-history traits in homozygotes very difficult. Nevertheless, several successful genetic analyses of aging in Drosophila suggest that with careful design, fruitful analysis of induced mutants affecting life span is possible. In the nematode Caenorhabditis elegans, mutations in the age-1 gene result in a life extension of some 70%; thus age-1 clearly specifies a process involved in organismic senescence. This gene maps to chromosome II, well separated from a locus (fer-15) which is responsible for a large fertility deficit in the original stocks. There is no trade-off between either rate of development or fertility versus life span associated with the age-1 mutation. Transgenic analyses confirm that the fertility deficit can be corrected by a wild-type fer-15 transformant (transgene); however, the life span of these transformed stocks is affected by the transgenic array in an unpredictable fashion. The molecular nature of the age-1 gene remains unknown and we continue in our efforts to clone the gene.