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.     

Lack of hypergravity-associated longevity extension in Drosophila melanogaster flies overexpressing hsp70

Previous studies have shown that Drosophila melanogaster males live longer if exposed to hypergravity at young age. Similarly, thermotolerance and climbing activity in old age are enhanced and it has been hypothetized that the 70 kDa inducible heat-shock protein (hsp70) may play a role in some of these effects. In this article, longevity, thermotolerance and climbing activity were studied in transgenic flies (OE+strain) over expressing hsp70 and in control flies harboring the transfection vector at the same chromosomal location but no extra-copies of the hsp70 gene (OE−strain). No positive effect from exposure to hypergravity at a young age was observed for longevity and climbing activity. Thermotolerance was increased by hypergravity but to the same extent in OE+ and OE− strains. Since no positive effect from hypergravity exposure or similar effects were observed in the two strains, it may be concluded that over expression ofhsp70 does not provide an advantage to flies subjected to a mild stress and that it may not be of any help during aging. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lifespan extension of Drosophila melanogaster through hormesis by repeated mild heat stress

We assessed the impact of repeated episodes of a mild heat stress on lifespan, fecundity, heat stress resistance and Hsp70 expression in Drosophila melanogaster. There was a significant increase in lifespan of females repeatedly exposed to a mild heat stress when measured in both a pair and a group situation. There was no effect on fecundity when the flies were first exposed to the mild heat stress at an age later than 3 days old, but when it did occur on day 3, there was a significant effect on cumulative fecundity levels over 18 days. The negative fitness effect appears to be the result of a direct cessation or reduction of oviposition during the first bout of stress exposure, and is influenced by the age at which this first exposure occurs. The mild heat stress had no impacton egg viability. The mild heat stress exposures increased resistance to potentially lethal heat stress and levels of Hsp70 expression in heat-exposed flies were higher than those in controls. This revised version was published online in July 2006 with corrections to the Cover Date.     

Aging results in an unusual expression of Drosophila heat shock proteins.

We used high-resolution two-dimensional polyacrylamide gel electrophoresis to evaluate the effect of aging on the heat shock response in Drosophila melanogaster. Although the aging process is not well understood at the molecular level, recent observations suggest that quantitative changes in gene expression occur as these fruit flies approach senescence. Such genetic alterations are in accord with our present data, which clearly show marked differences in the synthesis of heat shock proteins between young and old fruit flies. In 10-day-old flies, a heat shock of 20 min results in the expression of 14 new proteins as detectable by two-dimensional electrophoresis of [35S]methionine-labeled polypeptides, whereas identical treatment of 45-day-old flies leads to the expression of at least 50 new or highly up-regulated proteins. In addition, there is also a concomitant increase in the rate of synthesis of a number of the normal proteins in the older animals. Microdensitometric determinations of the low molecular weight heat shock polypeptides on autoradiographs of five age groups revealed that their maximum expression occurs at 47 days for a population of flies with a mean life span of 33.7 days. Moreover, a heat shock effect similar to that observed in senescent flies occurs in young flies fed canavanine, an arginine analogue, before heat shock.     

Effects of mild heat shocks at young age on aging and longevity in Drosophila melanogaster

Young adult flies were submitted to heat shocks (37°C) ofvarious durations (5, 10, 20, 40 or 60 min daily) for 1, 2 or 3 weeks. Aslight longevity increase, in both sexes, was only observed with thelowest heat shock. Longer shocks had neutral or negative effects. Fliessubmitted to the procedure providing a longevity increase did not show adelayed behavioral aging but survived longer at 37°C thancontrol flies. This higher thermotolerance was not associated with anincreased hsp70 induction. The results are discussed in connection withhormesis and previous results showing that hypergravity, an other mildstress, increases longevity and delays behavioral aging: different mildstresses may have contrasting effects on aging and longevity.     

Urocortin increases the expression of heat shock protein 90 in rat cardiac myocytes in a MEK1/2-dependent manner

We have previously demonstrated that urocortin protects cultured cardiac myocytes from ischaemic and reoxygenation injury and decreases the infarct size in the rat heart exposed to regional ischaemia and reperfusion. Urocortin-mediated cardioprotection is via activation of the mitogen-activated protein kinase (MAP kinase, MEK1/2) pathway. In addition, it is well documented that heat shock protein (hsp) 70 and hsp90 are cardioprotective against lethal stress. In this study we show, for the first time, that urocortin induces the expression of hsp90 but not hsp70 in primary cultures of rat neonatal cardiac myocytes. Levels of hsp90 protein increase by 1.5-fold over untreated cells within 10 min of urocortin treatment and are sustained for 24 h with a maximal increase of 2.5-fold at 60 min (P<0.05 at all time points). The increase in hsp90 expression by urocortin was not inhibited by actinomycin D, and urocortin failed to increase hsp90 promoter activity. Urocortin induction of hsp90 was inhibited by the MEK1/2 inhibitor PD98059 (P<0.001) and by cycloheximide, and both inhibitors abrogate urocortin-mediated cardioprotection (P<0.05 for cycloheximide, P<0.001 for PD98059). Hence, MEK1/2 and protein synthesis are involved in the cardioprotective effect of urocortin against hypoxic-mediated cell death, possibly due to an increase in expression of hsp90 protein. This is the first report of heat shock protein induction by urocortin or any other member of the corticotrophin-releasing hormone family.     

Male <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> flies exposed to hypergravity at young age are protected against a non-lethal heat shock at middle age but not against behavioral impairments due to this shock

Previous studies have shown that exposing flies to hypergravity (3 or 5g) for two weeks at young age slightly increases longevity of male flies and survival time at 37 °C of both sexes, and delays an age-linked behavioral change. The present experiments tested whether hypergravity could also protect flies from a non-lethal 37 °C heat shock applied at young, middle or old age (2, 4 or 6weeks of age). Various durations of exposure at 37 °C had similar deleterious effects on climbing activity, spontaneous locomotor activity and learning in flies that lived or not in hypergravity at young age. Therefore, hypergravity does not protect the behavior of flies from a deleterious non-lethal heat shock. Hypergravity increased longevity of virgin males and decreased that of mated ones; it also increased longevity of virgins at 25 °C, the usual rearing temperature, but not at 30 °C. Thus, the positive effect of hypergravity on longevity is observed only if flies are not subjected to living conditions decreasing longevity, like mating and high temperature. Finally, 4 weeks-old males that lived in hypergravity at young age lived slightly longer (+ 15%) after a non-lethal heat shock (60 or 90 min at 37 °C) than flies that always lived at 1 g, but this positive effect of hypergravity was not observed in females or in older males. Therefore, all these results show that hypergravity exposure can help male middle-aged flies recovering from a heat shock, but does not protect them from behavioral impairments linked to this shock: a mild stress occurring at young age can partially protect from a moderate stress at middle age.     

Similar gene expression patterns characterize aging and oxidative stress in Drosophila melanogaster

Affymetrix GeneChips were used to measure RNA abundance for approximately 13,500 Drosophila genes in young, old, and 100% oxygen-stressed flies. Data were analyzed by using a recently developed background correction algorithm and a robust multichip model-based statistical analysis that dramatically increased the ability to identify changes in gene expression. Aging and oxidative stress responses shared the up-regulation of purine biosynthesis, heat shock protein, antioxidant, and innate immune response genes. Results were confirmed by using Northerns and transgenic reporters. Immune response gene promoters linked to GFP allowed longitudinal assay of gene expression during aging in individual flies. Immune reporter expression in young flies was partially predictive of remaining life span, suggesting their potential as biomonitors of aging.     

Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene.

The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. We have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, two-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45 degrees C and their thermal survivals were determined by colony-formation assay, we found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress.     

An evolutionary heterogeneity model of late-life fecundity in <Emphasis Type="Italic">Drosophila</Emphasis>

There is now a significant body of research that establishes the deceleration of mortality rates in late life and their ultimate leveling off on a late-life plateau. Natural selection has been offered as one mechanism responsible for these plateaus. The force of natural selection should also exert such effects on female fecundity. We have already developed a model of female fecundity in late life that incorporates the general predictions of the evolutionary model. The original evolutionary model predicts a decline in fecundity from a peak in early life, followed by a plateau with non-zero fecundity in late life. However, in Drosophila there is also a well-defined decline in fecundity among dying flies, here called the “death spiral”. This effect produces heterogeneity between dying and non-dying flies. Here a hybrid evolutionary heterogeneity model is developed to accommodate both the evolutionary plateau prediction and the death spiral. It is shown that this evolutionary heterogeneity model gives a much better fit to late-life fecundity data.     

Heat shock protein-56 is induced by cardiotrophin-1 and mediates its hypertrophic effect.

Cardiotrophin-1 (CT-1) is an interleukin-6 family cytokine with known protective and hypertrophic effects in the heart. Previous studies have shown that CT-1 treatment increases heat shock protein 70 (hsp70) and heat shock protein 90 (hsp90) levels in cardiac cells. Due to the known protective effects of hsp90 and hsp70, induction of these proteins may be involved in the protective effects of CT-1.We show here that heat shock protein 56 (hsp56), also known as FK506 binding protein 59 (FKBP59), is induced by CT-1 treatment at both the mRNA and protein levels. It has been demonstrated previously that, unlike hsp70 and hsp90, hsp56 overexpression does not protect cardiac myocytes against stressful stimuli. The other known effect of CT-1 is hypertrophy, an increase in cell size without cell division, which occurs in many cardiac pathologies. We investigated the role of hsp56 in the hypertrophic response of primary neonatal rat cardiac myocytes, using overexpression with transiently transfected plasmid vectors and Herpes viral vectors. Overexpression of hsp56 caused a significant increase in cardiac cell size and protein:DNA ratio. Hsp27, hsp70 and hsp90 overexpression had no effect on cell size. An antisense construct to hsp56 reduced hsp56 levels when transiently transfected and blocked the hypertrophic effect of CT-1. This is the first time that a hypertrophic effect has been demonstrated for a heat shock protein and demonstrates that CT-1-induced hypertrophy involves a specific hsp, which is not involved in its protective effect.     

Longevity for free? Increased reproduction with limited trade-offs in Drosophila melanogaster selected for increased life span

Selection for increased life span in Drosophila melanogaster has been shown to correlate with decreased early fecundity and increased fecundity later in life. This phenomenon has been ascribed to the existence of trade-offs in which limited resources can be invested in either somatic maintenance or reproduction. In our longevity selection lines, we did not find such a trade-off. Rather, we find that females have similar or higher fecundity throughout life compared to non-selected controls. To determine whether increased longevity affects responses in other traits, we looked at several stress resistance traits (chill coma recovery, heat knockdown, desiccation and starvation), geotactic behaviour, egg-to-adult viability, body size, developmental time as well as metabolic rate. Longevity selected flies were more starvation resistant. However, in females longevity and fecundity were not negatively correlated with the other traits assayed. Males from longevity selected lines were slower at recovering from a chill induced coma and resting metabolic rate increased with age, but did not correlate with life span.     

Proteins related to the mouse L-cell major heat shock protein are synthesized in the absence of heat shock gene expression.

Heat shock of mouse L cells induces the synthesis of two polypeptides of Mrs 68,000 and 89,000. Using a fragment of a cloned gene encoding the Drosophila melanogaster Mr 70,000 heat shock protein (hsp70), we have shown that this protein has been highly conserved during eukaryotic evolution. We extended this observation by probing at low stringency for the expression in mouse L cells of RNA homologous to the Drosophila hsp70 gene. In addition to the RNA encoding the inducible Mr 68,000 heat shock protein (hsp68), there are mouse mRNAs encoding proteins of Mrs 70,000 and 74,000 that are homologous to the Drosophila hsp70 gene. The Mrs 70,000 and 74,000 proteins and their mRNAs are abundant components of unstressed mouse L cells. These constituitively expressed proteins are unique polypeptides in contrast to the several isoelectric point variants of the inducible hsp68. We do not detect hsp68 or its mRNA in unstressed L cells. In addition to the mRNAs corresponding to hsp68 and the Mrs 74,000 and 70,000 proteins, we detect a fourth RNA homologous to the Drosophila hsp70 gene but whose protein product has not been identified. Our results suggest that the hsp68 gene of mouse L cells is a member of a multigene family and that the individual family members are distinguishable by their degree of similarity but show differences in the regulation of their expression.     

Cold stress increases resistance to fungal infection throughout life in Drosophila melanogaster

Flies were subjected to one of three mild stresses known to have positive effects on longevity (heat, hypergravity, cold), prior to an infection with the entomopathogenic fungus Beauveria bassiana. Flies subjected to cold survived longer to infection, while the other mild stresses had no positive effect. These positive effects of a cold stress on resistance to infection were observed mainly in males and throughout life, i.e., a long time after the cold stress was applied. It was confirmed that cold and hypergravity stresses increased longevity of non-infected flies, but no positive effect of heat shocks were however observed.