Report

Extended Life-Span and Stress Resistance in the Drosophila Mutant methuselah

See allHide authors and affiliations

Science  30 Oct 1998:
Vol. 282, Issue 5390, pp. 943-946
DOI: 10.1126/science.282.5390.943

Abstract

Toward a genetic dissection of the processes involved in aging, a screen for gene mutations that extend life-span in Drosophila melanogaster was performed. The mutant line methuselah(mth) displayed approximately 35 percent increase in average life-span and enhanced resistance to various forms of stress, including starvation, high temperature, and dietary paraquat, a free-radical generator. The mth gene predicted a protein with homology to several guanosine triphosphate–binding protein–coupled seven–transmembrane domain receptors. Thus, the organism may use signal transduction pathways to modulate stress response and life-span.

The effect of genes on life-span in Drosophila has been established by selective breeding (1). However, the participation of multiple genes with additive, quantitative effects can be difficult to unravel. A direct search for life-extension mutants could identify individual genes that regulate biological aging. Indeed, in the nematodeCaenorhabditis elegans, several mutations, for example,age-1, daf-2, and clk-1, have been described that can increase the worm's life-span (2). The corresponding genes have been cloned and are involved in various aspects of development and metabolism (3,4).

Life-span and stress response are closely associated. In C. elegans, the age-1 mutant displays elevated resistance to thermal exposure (5) and to oxidative stress (6). In Drosophila, laboratory stocks selected for postponed senescence also show increased tolerance to heat, starvation, desiccation, and oxidative damage (7–9). Tandem overexpression of Cu-Zn superoxide dismutase (SOD) and catalase genes in Drosophila increased life-span by 30% (10). Similar observations were made in flies expressing the human SOD1 transgene in motor neurons (11). However, the physiological and molecular events involved in life-span determination and stress resistance have remained largely elusive.

We generated a set of P-element insertion lines (12,13) and screened them for ones that outlived a parent strain (white1118 ). methuselah(mth) was isolated by its increase in life-span at 29°C. The life extension was confirmed at 25°C. At both temperatures, flies homozygous for the P-element lived, on the average, 35% longer than the parent strain (Fig. 1).

Figure 1

Life-span extension inmethuselah. Male flies of the parental strain (white1118 ) and methuselah(homozygous for the P-element insertion) were maintained in a constant temperature, humidity, and 12/12 hour dark/light cycle environment. Flies were transferred to fresh food vials and scored for survival every 3 to 4 days. (A) Survival curve. The average life-spans forw1118 and mth were 57 and 77 days, respectively. The numbers of flies tested were 876 forw1118 and 783 for mth. (B) Mortality rate. Logarithm of mortality rate (the fraction of flies dying per day) is plotted against age.

We then examined the ability of mth flies to resist stress. mth mutant flies were more resistant to dietary paraquat (Fig. 2A), which, upon intake by the cell, generates superoxide anion (14). At a concentration of 20 mM, paraquat rendered normal males sluggish by 12 hours; at 48 hours, nearly 90% were dead. In contrast, mthmales were still active at 24 hours, and at 48 hours more than 50% were still alive. In a long-lived strain of Drosophiladerived by selection, life-span extension also accompanies increased paraquat resistance (9). Transgenic Drosophilacarrying extra copies of SOD and catalase, two primary components of the defense system against reactive oxygen species, also have increased life-span (10). Flies transgenic for the human SOD1 gene display increased life-span and paraquat resistance, the degree of effect correlating with dosage of the transgene (11). Thus,mth may have a higher capacity of the free-radical defense system.

Figure 2

Stress responses. Homozygous mthcompared with the parent strain. Newly eclosed flies were sex-segregated, distributed 20 per vial, and maintained in fresh food vials for 2 to 5 days before testing. Genotypes and sexes are indicated. (A) Paraquat resistance. Flies (age 2 days) were starved for 6 hours, then transferred to vials (2.5 cm by 9.3 cm) containing two 2.4-cm glass-fiber filter circles (Whatman) wetted with 20 mM paraquat (Sigma) in 5% sucrose solution, and survival was scored at 25°C. The ingestion rates of mthand the parent strain were similar, as determined by14C-leucine and dye intake. (B) Starvation test. Flies (age 2 days) were transferred to vials containing filters moisturized with 0.2 ml of distilled water. Distilled water was added to keep the filters moist during the test. (C) Thermal stress test. Flies (age 5 days) were transferred to vials containing 1% agar in 5% sucrose solution, and maintained at 36°C.

In the starvation test, mth showed a greater than 50% increase in average survival time over the parent strain (Fig. 2B). Females were consistently more resistant than males, suggesting that their larger body weight may contribute to resistance. Indeed,mth males and females weighed 20 to 30% more than their w1118 counterparts. In aDrosophila stock selectively bred for postponed senescence, resistance to starvation and lipid content are higher than the baseline stock (7). In C. elegans, the mutant daf-2, which exhibits marked increase in longevity, has extensive fat accumulation when grown at 25°C, suggesting a coupling of its metabolism with longevity (4).

Next we tested exposure to high temperature (Fig. 2C). At 36°C,mth survived longer than the parent strain. Heat shock proteins, a class of molecular chaperones, are thought to counter stress-induced detrimental effects during aging (15). In a transgenic fly harboring 12 additional copies of the heat-inducible hsp70 gene, there was a positive correlation between life expectancy and elevated Hsp70 protein expression (16). Correspondingly, in daf-2 andage-1 mutant worms, resistance to thermal stress was higher than in control animals (5). The increased thermotolerance of mth may result from higher expression of heat shock proteins and related molecular chaperones.

By Southern (DNA) blot analysis of mth genomic DNA, we confirmed that mth carries a single P-element insertion in the genome (17). Genetic mapping indicated that it is inserted in the third chromosome. By crossing mth to flies harboring a transposase (18), we generated lines in which the P-element was precisely excised from the insertion site (as determined by polymerase chain reaction). Eight lines obtained in this manner had life-spans reverted to that of the parent strain, indicating that the phenotype in mth was specifically caused by the P-element insertion. The precise-excision strains were used as controls throughout the study; they behaved similarly to the parental strain in stress resistance as well.

Two other lines isolated had imprecise excisions of the P-element, resulting in deletion of DNA adjacent to the insertion site. Both of these lines, which likely represent null alleles of themth gene, displayed pre-adult lethality in homozygotes, suggesting that the gene also plays an essential role in development. Flies heterozygous for the P-element over an imprecise excision allele were more resistant to stress than those homozygous for the P-element, indicating that the mutation created by the P-element insertion is a hypomorphic allele. The P-element insertion in the third intron of the mth gene may reduce the level of gene expression by interfering with RNA splicing, without eliminating the gene function.

We cloned the full-length genomic and complementary DNA of themth gene (19) (Fig. 3). The cDNA encodes a single open reading frame (Fig. 3B). The predicted protein sequence has a leader peptide plus seven hydrophobic regions suggestive of transmembrane (TM) domains (Fig. 3C). A gapped Blast search (20) of this sequence showed homology to a variety of guanosine triphosphate–binding regulatory protein (G protein)–coupled receptors (GPCRs) (Fig. 3D). GPCR was also predicted by the Blocks Search program (21). The amino acid residues between TM5 and TM6, especially those near the transmembranes, are highly basic, a feature shared by many G protein–linked receptors, and in some cases these residues interact directly with G proteins (22). Homology was found mainly in the TM regions. The NH2-terminal segment preceding the first TM domain was not found to share homology with any known sequence, thus diminishing the overall homology scores. The mth gene appears to represent a previously unknown member of the seven-TM protein superfamily. It remains to be seen whether the unique NH2-terminal sequence is related to the regulation of the MTH protein, and what the identity of its ligand (or ligands) might be.

Figure 3

The mth gene. The full-length cDNA and its corresponding genomic segment. (A) Genomic DNA. Restriction enzyme sites: E, Eco RI; P, Pst I; Sa, Sac I; Sm, Sma I; X, Xba I. Boxes indicate exons; hatched boxes, the open reading frame. The P-element insertion site is indicated by an arrow. The plasmid rescue clones, 44P1 and 44E1, represent, respectively, upstream and downstream fragments relative to the P-element. The structure is based on the genomic sequence derived from P1 plasmid DS06692 of the BDGP. (B) Complementary DNA and protein sequence. Numbers are indicated at the left and right, respectively. The putative leader peptide sequence is in boldface; TM domains are underlined. The polyadenylation site is boxed. The sequence is derived from LD08316 of the BDGP. (C) Hydropathic profile of the conceptual MTH protein, analyzed by the Kyte-Doolittle algorithm (29). The seven hydrophobic regions (excluding the NH2-terminal, putative leader peptide) are designated. (D) Alignment of MTH with several known G protein–coupled receptors. MTH protein is aligned to partial sequences of human leukocyte surface antigen CD97 (hCD97, GenBank accession number P48960), rat α-latrotoxin receptor (rLR, U72487), and mouse epidermal growth factor module–containing receptor (mEMR-1, Q61549). Dark shading indicates identity, light shading similarity. The seven TM domains of MTH are indicated by lines above each row. Consensus amino acids are cited below; similarity is indicated by dots. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Because life-span and stress response are closely related, genetic screening by stress resistance provides an effective alternative to the much slower direct screening for lifetime (23). The ability of the mth fly to resist various kinds of stress is notable because there are likely to exist differences in pathways of response to individual forms of stress.

G protein–coupled receptors are involved in a remarkably diverse array of biological activities including neurotransmission, hormone physiology, drug response, and transduction of stimuli such as light and odorants (24). Our data suggest that MTH is a GPCR involved in stress response and biological aging. By regulating an associated G protein and thus its downstream pathway, the normalmth gene may maintain homeostasis and metabolism, playing a central role in modulating molecular events in response to stress. The pre-adult lethality of the null alleles demonstrates that at least some activity of the mth gene is essential for survival. When mutated, the intermediate level of expression of a hypomorphic allele might adjust response to stress in a way that is more favorable for survival, whereas full expression of the normal gene exceeds the optimum value. The delicate balance among the embryonic lethality of a null allele, enhanced longevity of a hypomorphic allele, and the normal wild phenotype suggests that the level of mth gene expression is an important component of the system controlling life-span. Investigation of the gene's function and associated pathways should lead to better understanding of mechanisms relevant to aging.

  • * To whom correspondence should be addressed. E-mail: benzer{at}caltech.edu

REFERENCES AND NOTES

View Abstract

Navigate This Article