Differential patterns of apoptosis in response to aging in Drosophila

Abstract

Several lines of evidence suggest that programmed cell death may play a role in the aging process and the age-related functional declines of multicellular organisms. To pave the way for the use of Drosophila to rigorously test this hypothesis in a genetic model organism, this work examines the pattern of apoptosis in the adult fly during aging. The analysis across the lifespan of caspase activity and DNA fragmentation shows that apoptosis occurs in adult flies at all ages and that it is linked to physiological age. The results establish that under normal conditions, fly aging is coupled with a lifelong gradual increase of apoptosis within muscle cells and an activation of apoptosis in fat cells of old flies. The nervous system does not show signs of apoptosis. These time- and tissue-specific changes indicate that aging influences the levels and the nature of the cells that commit to apoptosis. The comparison with the apoptotic response to starvation and oxidative stresses strongly suggests that the lifelong increase in flight and leg muscles results from the accumulation of oxidative damage associated with aging. This finding presents an attractive mechanism to account for the decline of locomotor functions and muscle loss in the elderly and opens the way for the genetic analysis of sarcopenia in Drosophila.

Fig. 1.

Expression of apoptosis-related genes with increasing age. Concentration represented as 1× = 520 ng RNA. (A) Example of RNA samples, with two extractions (a and b) performed on males aged to 20, 30, and 40 days. Marker sizes indicated at left of gel image (base pairs). (B and C) RT-PCR analysis of sets of two RNA extracts (a and b) taken from males at varying ages, shown in days, with expected band size indicated (base pairs) at left with arrow. (B) Size of genomic DNA fragment equals mRNA band, therefore negative controls performed as lack of initial 30 min reverse transcription step, indicated (–) after concentration. (C) Positive control genomic DNA band size indicated (base pairs) at right with arrow.

Fig. 2.

DEVD activity as a function of age. (A) Contribution of various body parts. DEVD activity was quantified in head, thorax, and abdomen of freshly eclosed (<2 h), 2, 10, 20, 30, 40, and 50 days w¹¹¹⁸ males (solid line) and females (broken line). Data are shown as mean ± SE (n = 3). (B) DEVD activity in different genetic backgrounds. Age is shown as percent of maximum lifespan for each genotype. Each point represents the mean of at least three samples. Caspase activity in both male and female thorax significantly increases with age.

Fig. 3.

DNA fragmentation with increasing age. Representative TUNEL images show DNA fragmentation in the thorax of males at age 20% (A and B), 40% (C and D), and 60% (E and F) of lifespan. The fly diagrams at the top indicate the position of the sagital section in the pictures below. Inset shows the corresponding labeled area (c, f, and lm) at higher magnification. c, cardia; f, fat; lm, longitudinal muscle; vc, ventral nerve cord.

Fig. 4.

DEVD activity in response to dry starvation and oxidative stresses. Newly emerged w¹¹¹⁸ outcrossed to Canton-S were sorted by gender and aged to 4 days at 25°C. Controls were fed 5% sucrose. After 20 h of dry starvation or 3 h of starvation followed by 17 h of exposure to 20 mM paraquat in 5% sucrose, the dead flies were counted and caspase activity was measured in both the thorax and abdomen of controls (open bars), paraquat-treated (black bars), and starved (gray bars) males and females that survived. Paraquat stress increases caspase activity in both male and female thorax. However, starvation significantly reduces caspase activity in both male and female abdomen. Data are shown as mean plus SE (n = 10 for each group). *, 0.01 < P < 0.05; **, P < 0.01 compared with control (t test).