High-quality life extension by the enzyme peptide methionine sulfoxide reductase

Abstract

Cumulative oxidative damages to cell constituents are considered to contribute to aging and age-related diseases. The enzyme peptide methionine sulfoxide reductase A (MSRA) catalyzes the repair of oxidized methionine in proteins by reducing methionine sulfoxide back to methionine. However, whether MSRA plays a role in the aging process is poorly understood. Here we report that overexpression of the msrA gene predominantly in the nervous system markedly extends the lifespan of the fruit fly Drosophila. The MSRA transgenic animals are more resistant to paraquat-induced oxidative stress, and the onset of senescence-induced decline in the general activity level and reproductive capacity is delayed markedly. The results suggest that oxidative damage is an important determinant of lifespan, and MSRA may be important in increasing the lifespan in other organisms including humans.

Figure 1

Pan-neuronal MSRA overexpression and lifespan extension. (A) RT-PCR analysis. Lane 0, molecular weight markers; lane 1, elav-GAL4/Y adult male (parental control); lane 2, UAS-MSRA^C/UAS-MSRA^C (MSRA^C) adult male (parental control); lane 3, elav-GAL4/Y;UAS-MSRA^C/+ adult male; lane 4, elav-GAL4/+; UAS-MSRA^C/+ adult female; lane 5, elav-GAL4; UAS-MSRA^C/+ male and female larvae. The expected product size is 1.4 kb. EGFP-MSRA signals are present in lanes 3, 4, and 5. The adult animals used were 5–8 days old. Expression of EGFP-MSRA in the nervous systems was achieved by crossing the MSRA^C line and the elav^C155-GAL4 activator line. (B) Fluorescent micrographs of larval brain/ventral ganglion complexes. EGFP fluorescence was clearly observed in the elav-GAL4; UAS-MSRA^C/+ larval nervous system (Right), but only weak signals were present in the parental control larval MSRA^c nervous system (Left), indicating that the non-GAL4-activated EGFP-MSRA expression was negligible. A third instar larva was dissected to expose the nervous system and observed by using an Olympus BX51 fluorescence microscope with a GFP filter. (Scale bars, 200 μm.) E, eye-antennal disk; B, brain hemisphere; V, ventral ganglia. (C) Fluorescent micrographs of dissected adult brains. EGFP fluorescence was very faint in the parental control MSRA^C nervous system at the age of ≈8 days (Left) but clearly visible in the elav-GAL4; UAS-MSRA^C/+ adult nervous system at the age of ≈8 days (Center) and also at 70 days (Right). Adult brains were dissected out in PBS, mounted in 50% glycerol/PBS, and viewed with an Olympus BX51 microscope with a GFP filter. Images were digitized with a Spot RT Slider digital camera (Diagnostic Instruments, Sterling Heights, MI) with monochrome settings and then pseudocolored. (Scale bars, 50 μm.) O, optic lobe; B, central brain. (D) Survivorship curves of progeny animals resulting from crosses between MSRA^C and wild-type Oregon R (UAS-MSRA^C/+), elav-GAL4 and Oregon R (elav-GAL4/+), and elav-GAL4 and MSRA^C (elav-GAL4; UAS-MSRA^C/+) shown in two different representations. UAS-MSRA^C/+ (▿) and elav-GAL4/+ (○) represent the control groups, and MSRA is overexpressed in elav-GAL4; UAS-MSRA^C/+ (●). The estimated median lifespan values of the female animals in these groups were 55, 61, and 95 days, respectively. The median values for the male animals were 42, 48, and 77 days. The smooth lines represent best fits of the data using the two-parameter Gompertz survivor distribution S(t) = exp{(R₀/α)⋅[1 − exp(α⋅t)]} where t is adult age in days, and R₀ and α are the Gompertz parameters. The values of R₀ and α for UAS-MSRA^C/+, elav-GAL4/Y, and elav-GAL4/Y; UAS-MSRA^C/+ animals were 1.40 × 10⁻³/7.42 × 10⁻², 4.00 × 10⁻³/5.64 × 10⁻³, and 5.13 × 10⁻⁵/9.29 × 10⁻² for males and 4.25 × 10⁻⁴/7.95 × 10⁻², 1.52 × 10⁻³/6.03 × 10⁻², and 1.65 × 10⁻⁵/8.65 × 10⁻² for females. Similar results were obtained in another trial and also when MSRA^D/MSRA^D, elav-GAL4/+, and elav-GAL4; UAS-MSRA^D/+ were compared. Each group initially contained 60 animals, and each vial contained 20 male or female animals. (E) Survivorship curves of the parental control animals, elav-GAL4 and MSRA^C and elav-GAL4; UAS-MSRA^C/+ progeny that overexpresses MSRA in the nervous systems. The parental control lines elav-GAL4/elav-GAL4 (or elav-GAL4/Y, □) and MSRA^C/MSRA^C (○) shown were tested as homozygotes in Canton S and Oregon R genetic backgrounds, respectively. The estimated median lifespan values of elav-GAL4 and MSRA^C were 40 and 35 days for male animals and 46 and 39 days for female animals. The median lifespans for the male and female elav-GAL4;UAS-MSRA^C/+ (●) animals were 69 and 75 days. The smooth lines represent best fits of the data using the Gompertz distribution. The values of R₀ and α for MSRA^C and elav-GAL4/Y and elav-GAL4; UAS-MSRA^C/+ animals were 5.46 × 10⁻³/5.89 × 10⁻², 1.28 × 10⁻³/9.77 × 10⁻³ and 9.55 × 10⁻⁵/9.56 × 10⁻² for males, and 4.01 × 10⁻³/6.15 × 10⁻², 1.15 × 10⁻³/8.19 × 10⁻³, and 1.20 × 10⁻⁵/1.18 × 10⁻¹ for females, respectively. Each curve was based on 70 animals raised in vials, each containing 10 male and 10 female animals.

Figure 2

Survivorship and MSRA expression driven by Ubi and GMR. (A and B) Fluorescent micrographs of the Ubi-GAL4/+; UAS-MSRA^C/+ and GMR-GAL4/+; UAS-MSRA^C/+ larvae. GFP fluorescence was detected diffusely in the whole body, especially in muscle fibers of Ubi-GAL4/+; UAS-MSRA^C/+ (A) and primarily confined to the eye area of the eye-antennal discs of GMR-GAL4/+; UAS-MSRA^C/+ (B). (Scale bar, 100 μm.) O, optic lobe; B, brain hemisphere; V, ventral ganglia; E, eye-antennal disk; M, muscle fiber. Ectopic expression of EGFP-MSRA was achieved by crossing MSRA^C with the homozygous Ubi-GAL4 or GMR-GAL4 activator line. (C) Survivorship curves of Ubi-GAL4/+; UAS-MSRA^C/+ (●) compared with the parental lines Ubi-GAL4 (□) and UAS-MSRA^C (○). The smooth lines represent best fits of the data using the Gompertz survivor function. The estimated lifespan values for the male and female animals in the Ubi-GAL4 group were 45 and 51 days, respectively. The values in the Ubi-GAL4/+; UAS-MSRA^C/+ were 64 and 70 days. The values of R₀ and α for MSRA^C, Ubi-GAL4/Ubi-GAL4 and Ubi-GAL4/+; UAS-MSRA^C/+ animals were 5.46 × 10⁻³/5.89 × 10⁻², 1.10 × 10⁻³/8.73 × 10⁻², and 2.08 × 10⁻⁵/1.29 × 10⁻² for males and 4.01 × 10⁻³/6.15 × 10⁻², 3.28 × 10⁻⁴/1.05 × 10⁻¹, and 1.57 × 10⁻⁴/8.37 × 10⁻³ for females. (D) Survivorship curves of GMR-GAL4/+; MSRA^C/+ (●) compared with the parental lines GMR-GAL4 (□) and UAS-MSRA^C (○). The smooth lines represent best fits of the data using the Gompertz survivor function. The estimated lifespan values for the male and female animals in the GMR-GAL4 group were 45 and 51 days, respectively. The values in the GMR-GAL4/+; MSRA^C/+ were 64 and 70 days. The values of R₀ and α for MSRA^C/MSRA^C, GMR-GAL4/GMR-GAL4, and GMR-GAL4/+; MSRA^C/+ animals were 5.46 × 10⁻³/5.89 × 10⁻², 3.78 × 10⁻⁴/1.23 × 10⁻², and 4.60 × 10⁻⁴/9.53 × 10⁻² for males and 4.01 × 10⁻³/6.15 × 10⁻², 1.65 × 10⁻³/6.77 × 10⁻², and 6.50 × 10⁻⁴/8.32 × 10⁻² for females. The control data in C and D are the same.

Figure 3

Increased paraquat resistance by MSRA overexpression. (A) Paraquat resistance of 30-day-old male animals. In each trial, the experimental and control groups each consisted of 60 animals, and the number of animals alive after feeding for 24 h with paraquat was recorded. Each data point represents an average of 3–6 trials with medium containing the dye Brilliant Blue. Similar results were obtained when the dye was omitted. (B) Paraquat-containing food intake measurements on 30-day-old male animals. The average food intake of elav-GAL4/Y; UAS-MSRA^C/+ progeny was similar to that of elav-GAL4/Y and slightly greater than that of MSRA^C animals. Intake of paraquat-sucrose solution was measured by using the dye Brilliant Blue as described for A.

Figure 4

MSRA overexpression delays senescence-induced declines in physical activity and reproductive levels. (A) Changes in the general activity level with age. The “active fraction” is defined as the fraction of animals on the wall after vortexing the vial. Each data point represents an average of 3–6 determinations, each of which involved 20–40 male flies. Separate groups of animals were tested at different ages. At a given age, the animals overexpressing MSRA predominantly in the nervous system (elav-GAL4/Y; UAS-MSRA^C/+ progeny, ●) were more active than the parental control groups elav-GAL4/Y (□) and MSRA^C (○). (B) Changes in pupa production with age. The same groups of adult animals were transferred to fresh vials at fixed intervals as in the lifespan experiments, and the total number of the pupal progeny in each vial was counted. The results from the control groups elav-GAL4/Y (□) and MSRA^C (○) and the pan-neuronal MSRA-overexpression group elav-GAL4/Y; UAS-MSRA^C/+ (●) are shown. The ordinate represents the number of pupae produced per day per animal in a vial. Each data point represents results from four vials. In all the groups, >95% of the pupae developed into adults. (C) Copulation probabilities in male animals of different genotypes. Twenty adult males (OR, elav-GAL4/Y, MSRA^C, elav-GAL4/Y;UAS-MSRA^C/+) of 4–5 days of age were placed with 20 virgin wild-type CS females (6–7 days old) in a 1% agar-plated Petri dish (10-cm diameter). The number of animal pairs copulating was counted every 3 min and normalized to obtain the probability values. Each data point represents an average of 2–4 determinations.