Phosphatidylethanolamine positively regulates autophagy and longevity.

Autophagy is a cellular recycling program that retards ageing by efficiently eliminating damaged and potentially harmful organelles and intracellular protein aggregates. Here, we show that the abundance of phosphatidylethanolamine (PE) positively regulates autophagy. Reduction of intracellular PE levels by knocking out either of the two yeast phosphatidylserine decarboxylases (PSD) accelerated chronological ageing-associated production of reactive oxygen species and death. Conversely, the artificial increase of intracellular PE levels, by provision of its precursor ethanolamine or by overexpression of the PE-generating enzyme Psd1, significantly increased autophagic flux, both in yeast and in mammalian cell culture. Importantly administration of ethanolamine was sufficient to extend the lifespan of yeast (Saccharomyces cerevisiae), mammalian cells (U2OS, H4) and flies (Drosophila melanogaster). We thus postulate that the availability of PE may constitute a bottleneck for functional autophagy and that organismal life or healthspan could be positively influenced by the consumption of ethanolamine-rich food.

Spermidine promotes stress resistance in Drosophila melanogaster through autophagy-dependent and -independent pathways.

The naturally occurring polyamine spermidine (Spd) has recently been shown to promote longevity across species in an autophagy-dependent manner. Here, we demonstrate that Spd improves both survival and locomotor activity of the fruit fly Drosophila melanogaster upon exposure to the superoxide generator and neurotoxic agent paraquat. Although survival to a high paraquat concentration (20 mM) was specifically increased in female flies only, locomotor activity and survival could be rescued in both male and female animals when exposed to lower paraquat levels (5 mM). These effects are dependent on the autophagic machinery, as Spd failed to confer resistance to paraquat-induced toxicity and locomotor impairment in flies deleted for the essential autophagic regulator ATG7 (autophagy-related gene 7). Spd treatment did also protect against mild doses of another oxidative stressor, hydrogen peroxide, but in this case in an autophagy-independent manner. Altogether, this study establishes that the protective effects of Spd can be exerted through different pathways that depending on the oxidative stress scenario do or do not involve autophagy.

Chronological aging-independent replicative life span regulation by Msn2/Msn4 and Sod2 in Saccharomyces cerevisiae.

Mutations in RAS2, CYR1, and SCH9 extend the chronological life span in Saccharomyces cerevisiae by activating stress-resistance transcription factors and mitochondrial superoxide dismutase (Sod2). Here we show that mutations in CYR1 and SCH9 also extend the replicative life span of individual yeast mother cells. However, the triple deletion of stress-resistance genes MSN2/MSN4 and RIM15, which causes a major decrease in chronological life span, extends replicative life span. Similarly, the overexpression of superoxide dismutases, which extends chronological survival, shortens the replicative life span and prevents budding in 30-40% of virgin mother cells. These results suggest that stress-resistance transcription factors Msn2/Msn4 negatively regulate budding and the replicative life span in part by increasing SOD2 expression. The role of superoxide dismutases and of other stress-resistance proteins in extending the chronological life span of yeast, worms, and flies indicates that the negative effect of Sod2, Msn2/Msn4/Rim15 on the replicative life span of S. cerevisiae is independent of aging.

Resistance to stress as a function of age in transgenic Drosophila melanogaster overexpressing Hsp70.

This study investigated the resistance to stress as a function of age in Drosophila melanogaster overexpressing Hsp70. The resistances to starvation, paraquat, and cold in flies from 1 to 7 week-old have been measured. The line carrying the insertion vector without the transgenes is more resistant to starvation and cold than the parental and transgenic lines. In contrast, transgenic flies carrying extra-copies of hsp70 are more resistant to paraquat, however this is due to an especially high resistance in two age groups compared to all the other groups. I showed that exposure to a mild heat shock does not increase starvation resistance, slightly increases paraquat resistance, and strongly increases cold resistance. The transgenic flies expressing Hsp70 at higher levels after the heat shock do not exhibit enhanced stress resistance compared to control lines expressing less Hsp70 after the heat shock. The lack of effect of a mild heat shock on starvation and paraquat resistance is not due to a disappearance of the effect with age, since no effect is observed at any age. In contrast, when an effect of Hsp70 induction is observed as on cold resistance, this effect is still observed in old flies.

Locomotor activity as a function of age and life span in Drosophila melanogaster overexpressing hsp70.

Heat shock protein induction might be responsible for the longevity increase conferred by exposure to non-lethal stresses. To test this hypothesis, we studied in transgenic Drosophila melanogaster overexpressing hsp70 and controls, two behavioral variables (spontaneous locomotor activity and climbing activity) to evaluate the rate of aging, and life span. The results showed that in flies kept in groups, life span was decreased in transgenic flies compared to the parental line, but the contrary was observed in individually kept flies. Hsp70 overexpression had no dramatic effect on life span. Furthermore, we did not detect any advantage of Drosophila overexpressing hsp70 on the two measurements of locomotor activity. These results indicate that the rate of aging in transgenic flies is not different than in non-transgenic lines and that they are not more able to cope with the effects of aging on locomotor activity.

Longevity and aging: beneficial effects of exposure to mild stress.

Every organism has to deal with exposure to stresses. Animals have developed various strategies to cope with stress. It appears that the developed resistance to stress is often related to longevity. Some scientists have advanced the hypothesis that the stress response may also counteract the negative effects of aging, and that exposing organisms to a mild, sublethal stress, inducing a stress response, may help them to live longer. Several mild stresses have been reported to increase longevity (irradiation, heat and cold shock, hypergravity, exercise, etc.), and one of them, hypergravity, to decrease the rate of behavioral aging. The mechanisms whereby these stresses increase longevity have not yet been elucidated. However, the studies conducted so far show that they may involve metabolic regulation and stress protein (hsps) induction.

A mild stress due to hypergravity exposure at young age increases longevity in Drosophila melanogaster males.

Drosophila melanogaster flies were exposed to hypergravity starting at two days of age, the range of gravity levels used being 2.58-7.38 g. No longevity change was observed for exposures of less than 14 days. The longevity of males increased if they were submitted to hypergravity for durations ranging from 14 to 24 days. This increase in longevity was never observed in females. The positive effect of exposure to hypergravity has been replicated in two laboratories using two wild-type strains and different rearing conditions. A short hypergravity exposure seems to be a mild stress, yielding positive effects on longevity. This is in accordance with two previous studies showing a slight longevity increase after heat shock in the nematode Caenorhabditis elegans and in Drosophila melanogaster.

Resistance to stress as a function of age in Drosophila melanogaster living in hypergravity.

Male and female fruitflies (Drosophila melanogaster) living at different gravity levels [1g: terrestrial gravity; 3 and 5g: hypergravity (HG)] were used to investigate the age-specific (young: 7 days; middle-aged: 28 days; and old: 49 days) resistance to various stresses (starvation, desiccation, and cold). The experiment showed that the resistance of the flies to the studied stresses decreased with age, except in the case of females submitted to starvation which was increased. These variations were explained by the amount of lipid. Variation in desiccation resistance was not explained by the amount of water. As a function of gravity, no or slight differences were observed for the studied stresses. The resistance to heat of young flies increased with the gravity level. This resistance was not explained by a decreased locomotor activity of HG-living flies during heat stress, nor by the water and lipid contents.

HSP70 induction may explain the long-lasting resistance to heat of Drosophila melanogaster having lived in hypergravity.

In this study, we showed that in flies kept for 2 weeks at 1 (terrestrial gravity), 3 or 5 x g (hypergravity, HG) before transfer to 1 x g, resistance to heat remained higher in HG flies for several weeks after the transfer. The measurement of heat shock protein 70 (hsp70) indicated no induction of the protein in HG, but the study revealed that flies living in HG expressed more hsp70 only after being submitted to severe stress. The higher induction of hsp70 may explain the higher thermotolerance of these HG-treated young flies. Finally, an unknown protein was observed only in females. This protein may belong to a class of higher molecular weight hsp (hsp110), which have not previously been observed in Drosophila.

A mild stress, hypergravity exposure, postpones behavioral aging in Drosophila melanogaster.

Flies were submitted to two weeks of hypergravity in a centrifuge (3 or 5 g), starting at the second day of imaginal life, and their behavior (spontaneous locomotor activity, patterns of movement, and climbing activity) was observed from removal of the centrifuge to an older age; the usual effects of age on these behaviors were generally observed. Hypergravity-kept flies had worse behavioral scores on removal of centrifuge than those always kept at 1 g. When they aged, they got either similar or better scores than 1 g flies, which indicates that their behavioral aging may be slower. These results show that a mild stress such as hypergravity, which has been previously shown to increase the longevity of males and resistance to heat shock in both sexes, is an environmental manipulation postponing aging in flies.

Hypergravity and aging in Drosophila melanogaster. 9. Conditioned suppression and habituation of the proboscis extension response.

In a first experiment, the conditioned suppression of the proboscis extension response (PER) to sucrose was measured in young, middle-aged and old male Drosophila melanogaster flies living at either 1, 3 or 5 g. Flies were starved and then subjected to a learning task involving a sucrose stimulus, followed by an aversive one applied to their forelegs. In this learning task, flies learn to not extend their proboscis when walking on sucrose. Flies which have lived in hypergravity (HG) had a lower number of PER suppressions than 1 g ones, and this finding was mainly due to young and middle-aged flies. In a second experiment, the habituation of the PER was studied using as stimulation sucrose solutions 2-fold (first experiment), 4-fold (second one) or 8-fold (third one) higher than the individual sucrose threshold. Middle-aged and old flies habituated more slowly than young flies in the second and third experiments. In the third experiment, a decreasing speed of habituation was observed when gravity increased; this result was mainly due to young flies, and no gravity effect was observed in the other two age groups. This whole set of results suggests that HG-kept flies do not age faster than 1 g ones, as far as these learning and habituation tasks are concerned. It seems possible that HG acts like a mild stress to which flies adapt; if applied for a long time, HG could induce a premature aging, as observed in the previous papers of this series.

Increased longevity and resistance to heat shock in Drosophila melanogaster flies exposed to hypergravity.

In recent years, attempts have been made to increase longevity in animal models (caloric restriction in rodents or overexpression of catalase and superoxide dismutase in transgenic flies, for instance). We report here that flies submitted to hypergravity (3 or 5 g), for 1 or 4 weeks starting from the second day of imaginal life and transferred after that time to 1 g, have a higher resistance to heat shock than flies living continuously at 1 g. Furthermore, male flies that had lived for 2 weeks from the second day of life at 3 or 5 g, lived longer than those living all the time at 1 g; no longevity increase was observed in females. As far as we know, this is the first example in flies showing that a mild stress at a young age not only increases resistance to an acute stress but also increases longevity. A hypothesis to explain these results could be that heat-shock proteins, which are induced by various stress factors, are synthesized in conditions of hypergravity.

Failure to confirm increased longevity in Drosophila melanogaster submitted to a food restriction procedure.

Several studies have shown that, contrary to what occurs in rodents and in some invertebrate species, food restriction has no positive effect on longevity in Drosophila melanogaster. However, Chippindale et al. (1993) reported that flies subjected to food restriction, by modulating the yeast level, could live longer. In the present study we used the same yeast levels as Chippindale et al. in an attempt to confirm these results. No positive effect of food restriction on longevity could be observed in either sex in mated and virgin flies.