Induced Overexpression of Mitochondrial Mn-Superoxide Dismutase Extends the Life Span of Adult Drosophila melanogaster

Corresponding author: John Tower, Department of Biological Sciences, SHS 172, University of Southern California, University Park, Los Angeles, CA 90089-1340., jtower{at}USC.edu (E-mail)

Communicating editor: T. F. C. MACKAY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A transgenic system ("FLP-out") based on yeast FLP recombinase allowed induced overexpression of MnSOD enzyme in adult Drosophila melanogaster. With FLP-out a brief heat pulse (HP) of young, adult flies triggered the rearrangement and subsequent expression of a MnSOD transgene throughout the adult life span. Control (no HP) and overexpressing (HP) flies had identical genetic backgrounds. The amount of MnSOD enzyme overexpression achieved varied among six independent transgenic lines, with increases up to 75%. Life span was increased in proportion to the increase in enzyme. Mean life span was increased by an average of 16%, with some lines showing 30–33% increases. Maximum life span was increased by an average of 15%, with one line showing as much as 37% increase. Simultaneous overexpression of catalase with MnSOD had no added benefit, consistent with previous observations that catalase is present in excess in the adult fly with regard to life span. Cu/ZnSOD overexpression also increases mean and maximum life span. For both MnSOD and Cu/ZnSOD lines, increased life span was not associated with decreased metabolic activity, as measured by O₂ consumption.

CURRENT theory suggests that aging results from the decreased force of natural selection acting on older individuals (ROSE 1991 ; PARTRIDGE and BARTON 1993 ; CHARLESWORTH 1994 ; KIRKWOOD 1995 ; KIRKWOOD and AUSTAD 2000 ). Reduction in the force of natural selection with age allows each generation to inherit mutations that have deleterious effects at late ages. The accumulation of such mutations in the germ line results in reduced fitness of old organisms, including an imbalance between damage and repair and a failure to maintain somatic tissue structure and function.

Reactive oxygen species (ROS) are produced as a toxic byproduct of normal cellular metabolism, and oxidative damage is hypothesized to be one cause of aging in metazoans (HARMAN 1956 ; STADTMAN 1992 ; MARTIN et al. 1996 ; SOHAL and WEINDRUCH 1996 ; WALLACE 1999 ; FINKEL and HOLBROOK 2000 ). The enzymes catalase and superoxide dismutase (SOD) are major defenses against ROS in all cells. SOD exists in two forms inside the eukaryotic cell, Cu/ZnSOD primarily in the cytoplasm and MnSOD in the mitochondria. The major ROS produced by the cell is mitochondrial superoxide, which is converted by SOD to H₂O₂. Catalase converts H₂O₂ to molecular oxygen and water. These and other cellular defenses against ROS are not completely efficient. Oxidatively damaged macromolecules accumulate in every aging organism examined, and oxidative damage is implicated in the etiology of most human aging-related diseases.

Numerous studies demonstrate the correlation between aging and oxidative damage in Drosophila. In Drosophila, aging has been found to correlate with increased levels of protein carbonyls and 8-oxo-Guanine in the DNA (ORR and SOHAL 1994 ; SOHAL et al. 1995 ). Drosophila lacking either catalase or Cu/ZnSOD have reduced life span (MACKAY and BEWLEY 1989 ; PHILLIPS et al. 1989 ; ORR et al. 1992 ; GRISWOLD et al. 1993 ). Moreover, the stress response protein hsp70 is induced in muscle tissue during Drosophila aging and this expression is increased or accelerated in catalase and Cu/ZnSOD null mutant flies (WHEELER et al. 1995 , WHEELER et al. 1999 ). Finally, genetic selection of Drosophila populations for increased life span in the laboratory correlates with increased expression of oxidative stress resistance enzymes and increased oxidative stress resistance (ARKING et al. 1991 , ARKING et al. 2000 ).

One way to directly test the hypothesis that oxidative damage causes aging is to engineer transgenic organisms with increased oxidative stress defenses and determine the effects on life span. In pioneering experiments Drosophila were created that contained catalase and Cu/ZnSOD transgenes under the control of their native promoters or a constitutive heterologous promoter (SETO et al. 1990 ; REVEILLAUD et al. 1991 ; ORR and SOHAL 1992 , ORR and SOHAL 1993 , ORR and SOHAL 1994 ; GRISWOLD et al. 1993 ; SOHAL et al. 1995 ). While in certain cases increased stress resistance and life span were reported, current interpretation of the data suggests that increased life span was not demonstrated (TOWER 1996 ; KAISER et al. 1997 ; TATAR 1999 ). In contrast, when tissue-specific (GAL4/UAS; PARKES et al. 1998 ) or inducible (FLP-out; SUN and TOWER 1999 ) transgenic systems were used to cause overexpression of Cu/ZnSOD, it resulted in life span increases of up to 48%. Catalase overexpression increased the resistance of flies to H₂O₂ but had neutral or slightly negative effects on life span, both alone and in combination with Cu/ZnSOD overexpression (SUN and TOWER 1999 ). The data suggest that with regard to life span Cu/ZnSOD activity is limiting while catalase exists in excess. Consistent with this idea, analysis of multiple catalase mutant strains demonstrates that catalase activity must be reduced to < 10% of wild-type levels before reduced life span is observed (MACKAY and BEWLEY 1989 ; ORR et al. 1992 ; GRISWOLD et al. 1993 ).

Mitochondria are thought to be the primary cellular source of ROS and may also be primary targets of oxidative damage. Because of this, the exclusively mitochondrial form of superoxide dismutase, MnSOD, may be a particularly important additional defense against oxidative damage during aging. In mice MnSOD appears to be the more critical enzyme, as MnSOD mutations have more severe phenotypes than Cu/ZnSOD mutations (HUANG et al. 1999 ; WALLACE 1999 ). In this study Drosophila were engineered to overexpress mitochondrial MnSOD, using the FLP-out system to assay for effects on life span.

Environmental manipulations that decrease metabolic rate in Drosophila, e.g., isolation from mates (PARTRIDGE et al. 1987 ), dietary restriction (CHIPPINDALE et al. 1993 ), or decreased temperature (MIQUEL et al. 1976 ), also increase life span. To test whether SOD overexpression was enhancing life span through a dampening effect on metabolic rates, O₂ consumption was assayed throughout the life span of the flies overexpressing MnSOD or Cu/ZnSOD, as well as Oregon-R wild-type controls.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmid construction and transformation:
The 800-bp EcoRI fragment containing the full-length Drosophila melanogaster MnSOD cDNA was isolated from plasmid pcMnSOD (DUTTAROY et al. 1994 ) and substituted for the Cu/ZnSOD cDNA fragment in construct pActStopSOD (SUN and TOWER 1999 ) to generate construct mSOD. Six independent transgenic lines were generated for mSOD using published methods (RUBIN and SPRADLING 1982 ; SUN and TOWER 1999 ), and each was confirmed to contain a single P-element insertion using Southern analysis (data not shown). Each transgenic construct insertion chromosome was made homozygous by crossing to the same set of inbred balancer stocks. The names of all transgenic lines indicate the transgenic construct (e.g., mSOD), followed by the chromosome of insertion (X, 2, or 3), followed by a letter or number (or a letter/number combination) indicating the particular independent insertion on that chromosome. For example, mSOD2C is an insertion of the mSOD construct on the second chromosome, independent line "C," and CAT2A2 is an insertion of the previously reported catalase FLP-out construct CAT on the second chromosome, independent line "A2" (SUN and TOWER 1999 ).

General:
Drosophila culture, transformation, and life span assays were performed as previously described (SUN and TOWER 1999 ), with the modification that aging cohorts of flies consisted of 250–400 individuals for each of heat-pulsed (HP) and control (Co) groups, and were transferred to fresh food vials every 48 hr. The heat pulse regimen was as previously described for use with the FLP3 strain. All of the data presented here are novel and have not been previously reported. For strains that have previously been described, all data represent novel reassays of those strains and were essentially identical to previous results (SUN and TOWER 1999 ).

Drosophila culture, FLP-out heat pulse regimen and life span assays:
To obtain adult flies of defined age, the crosses described below were cultured at 25° in urine specimen bottles. Prior to eclosion of the majority of pupae, bottles were cleared of adults and newly eclosed flies were allowed to emerge over the next 48 hr. The majority of the males will have mated during this time. The males only were then removed, designated as 1 day old, maintained at 25° at 40/vial in culture vials with food, and passaged to new vials every 48 hr. At 5 days of age the males were pooled, separated into control and experimental groups of 250–400 flies each, and placed in culture vials with food at 40/vial. The control group was maintained at 25° and the experimental group was subjected to a heat pulse of 90 min at 37°. The experimental group was returned to 25° and then 24 hr later (day 6) the experimental group was subjected to a second heat pulse. The control and experimental groups were then maintained at 25° and passaged to fresh vials every 48 hr. Dead flies were counted at each passage, and the number of vials was progressively reduced to maintain approximately 40 flies/vial. To calculate mean life spans for the experimental and control groups, each fly's life span was tabulated, their life spans were averaged, and the SD and SEM were calculated. Maximum life span for the control and experimental cohorts was calculated as the amount of time in days required to reach 90% mortality. Life span was assayed at least twice and for several genotypes three times, using independently cultured flies in experiments separated in time by >2 weeks. Life span assays done simultaneously are indicated as Exp 1, Exp 2, Exp 3, and Exp 4 in the Tables.

SOD enzyme activity assays:
Cu/ZnSOD activity was measured as total SOD activity as previously described (SUN and TOWER 1999 ), except that EDTA concentration of assays was 0.08 mM. MnSOD activity was measured in the same way, except that assays contained 0.1 mM potassium cyanide to inhibit Cu/ZnSOD activity. SOD-specific activities are expressed as percentage inhibition of quercetin oxidation per microgram extract protein per 10-min reaction. Catalase-specific activity was measured as previously described (SUN and TOWER 1999 ) and is expressed in units of OD₂₄₀/min/µg extract protein. SOD and catalase activities are presented as the mean and standard deviation of triplicate extracts.

Metabolic rate assay:
Data for oxygen consumption experiments are presented in Table 4. The genotypes, culture, and heat pulse regimen were as described above. On day 6, following the second heat pulse, one-half of each cohort of SOD overexpressing flies was kept at University of Southern California in Los Angeles for the life span assays of Exp 3 and Exp 4, and one-half was moved to University of California-Irvine at controlled temperature for oxygen consumption assay. The oxygen consumption assay for the SOD overexpressing flies was therefore done on sibling flies of those used for life span assays Exp 3 and Exp 4. For the oxygen consumption assay, adult male flies from each treatment (control and HP) were divided into six replicate groups composed of 32–41 flies each and placed in maintenance vials containing food. Flies were transferred to new food vials every 2–3 days. Measurements of oxygen consumption were made using a flow-through respirometry system (Sable Systems, Henderson, NV) in a temperature-controlled room at 25° ± 1°. Flies were anesthetized with CO₂ and transferred from the maintenance vials into respirometry chambers (volume 10 ml) containing 0.5 ml food. Following recovery of the flies from anesthesia (20 min), dry, CO₂-free air was allowed to flow through the respirometry chambers at a rate of 6 ml/min, and oxygen decrement was measured using an Ametek (Pittsburgh, PA) S-3A/1 oxygen analyzer. Data were recorded and analyzed using Sable Systems' Datacan software. Metabolic rate was estimated using data from the final 10 min of the 30-min assay period to avoid the initial washout period.

The number of flies per chamber varied depending upon mortality, but initial numbers ranged from 32 to 41. Accurate estimates of metabolic rate/fly could be made within fly groups composed of as few as 5–10 flies each. The order in which flies were assayed for metabolic rate was varied systematically and experimental and control groups were measured in the same assay cycle. The food itself was found to have no measurable rate of oxygen uptake.

Oxygen consumption for all flies was measured once a week. Oxygen consumption in all Cu/ZnSOD lines and their controls was assayed over a 4-week period. MnSOD lines and their controls were assayed for 6–7 weeks, due to their increased longevity.

To estimate mean metabolic rate per fly, total oxygen consumption from each experimental chamber was divided by the total number of flies within each chamber and averaged over total number of weeks. Averages and SD were then calculated from the six replicate vials per treatment (Co and HP). These values were compared between control and experimental flies using two-sided t-tests and are presented in Table 3.

Statistical analyses: t-tests:
Mean life span and enzyme activities were compared between control and heat pulse populations using unpaired two-sided t-tests for each individual experiment. Maximum life span (time to 90% mortality) was compared between control and heat pulse populations by combining the paired data from two experiments using paired two-sided t-tests. Results are presented in Table 1 and Table 3.

ANOVA:

The following two standard ANOVA models were used (NEETER et al. 1990 ).

1. Two-factor fixed-effects ANOVA model:
   

   

   where
   

   

   _i are constants subject to the restriction _i = 0, ß_j are constants subject to the restriction ß_j = 0, (ß)_ij are constants subject to the restrictions
   

   

   _ijk are independent N(0, ²), i = 1, ... , a; j = 1, ... , b; k = 1, ... n. 2. Three-factor fixed effects ANOVA model:
   

   

   where
   

   

   _i, ß_j, _k, (ß)_ij, ()_ik, (ß)_jk, (ß)_ijk are constants subject to the restrictions
   

   

   i = 1, ... , a; j = 1, ... , b; k = 1, ... c; m = 1, ... , n.

The term "randomized block ANOVA" refers to an ANOVA design where experimental units within each block are randomly assigned treatment. Mathematically, a randomized block design with fixed blocks is equivalent to a fixed-effects ANOVA model where the blocks are treated as one of the fixed effects.

Induced expression of MnSOD transgene increases MnSOD enzyme activity:
To generate genotypes 1–6 of Table 1 and Table 3, strains homozygous for the indicated mSOD insertions were crossed to the FLP3 stock containing the hs:FLP construct on the third chromosome (w;FLP3 Sb/TM2 Ubx). Progeny were selected that inherited the FLP3 chromosome marked with the dominant mutation Sb and were therefore heterozygous for both constructs. The MnSOD enzyme activity data (Table 1) were analyzed using randomized-blocks ANOVA. The model for analysis was the two-factor fixed-effects ANOVA model. _i was the effect for genotype i, where i = 1, ... 6 (genotypes 1–6 of Table 1). ß_j was the effect for group (control vs. heat pulse) j, where j = 1, 2. There were three replications of the enzyme activity assay, k = 1, ... 3. The responses were denoted as Y_ijk, which indicates the activity level for genotype i, group j, and repetition k. The interaction between genotype i and group j was denoted as (ß)_ij. ANOVA table results are presented in Table 2.

Heat pulse alone does not increase MnSOD enzyme activity:
Genotypes 7–9 of Table 1 and Table 3 are controls that do not contain a SOD transgene. Genotype 7 is the progeny generated by crossing flies homozygous for the FLP-out lacZ expression construct on the third chromosome (SUN and TOWER 1999 ) to the FLP3 stock. Genotype 8 is the progeny generated by crossing the FLP3 stock to Oregon-R wild type. Genotype 9 is Oregon-R wild type. The data for the control genotypes 7–9 (Table 1) were analyzed using the two-factor fixed-effects ANOVA model as described above, where i = 7, 8, 9. Results are presented in the text.

Induced expression of MnSOD transgene increases mean and maximum life span:
Mean life span data for the MnSOD overexpressing genotypes 1–6 (Table 3) were analyzed using the two-factor fixed-effects ANOVA model as described above, except that the responses Y_ijk were life span, and there were two replications k = 1, 2. Maximum life span data for the MnSOD overexpressing genotypes 1–6 (Table 3) were analyzed using the same model.

Heat pulse alone does not increase life span:
Mean life span data for the control genotypes 7–9 (Table 3; i = 7, 8, 9) were analyzed using the two-factor fixed-effects ANOVA model as described above. Responses Y_ijk were life span, and there were two replications k = 1, 2. Maximum life span was analyzed in the same way.

Life span is increased in proportion to the increase in MnSOD enzyme activity:
The percentage increase in life span (mean and maximum) was plotted vs. the percentage increase in MnSOD enzyme activity (Fig 2), revealing a linear relationship for both. The data were analyzed using a standard linear regression model, namely, , where _i was normally distributed with mean 0 and variance ². The subscript i indicated genotype, where i = 1, ... 6. The life span for genotype i was denoted as Y_i, while the enzyme activity for genotype i was denoted as x_i. The intercept of the regression line was denoted as , while the slope was denoted as ß.

View larger version (34K): In this window In a new window Download PPT slide   Figure 1. Cumulative survival as a function of time for selected control and MnSOD overexpressing lines. The data are presented for life span assay, experiment 1, genotypes 1, 2, 4, 7, 8, and 9 of Table 3. Circles, control flies; squares, heat-pulsed flies. (A) mSODX1/Y;FLP3/+ genotype 1 of Table 3. (B) mSOD2C/+;FLP3/+ genotype 2 of Table 3. (C) mSOD2A/+;FLP3/+ genotype 4 of Table 3. (D) LacZ/FLP3 genotype 7 of Table 3. (E) FLP3/+ genotype 8 of Table 3. (F) Oregon-R genotype 9 of Table 3.

View larger version (19K): In this window In a new window Download PPT slide   Figure 2. Correlation between MnSOD overexpression and life span extension. For all six independent transgenic lines, the percentage increase in MnSOD enzyme activity was plotted vs. the percentage increase in mean life span (solid circles, solid line; r2 = 0.9375) and maximum life span (open squares, dashed line; r2 = 0.8283, 90% mortality); data are from Table 1 and Table 3.

Catalase overexpression is not associated with increased life span:
Genotype 10 is the progeny generated by crossing flies homozygous for the previously described catalase FLP-out expression construct on the second chromosome (CAT2A2; SUN and TOWER 1999 ) to the FLP3 stock. Genotype 11 was generated by crossing a strain homozygous for both the mSODX1 insertion and the CAT2A2 insertion to FLP3. Genotype 12 was generated by crossing a strain homozygous for both the CAT2A2 insertion and the mSOD3C insertion to FLP3. The data for genotypes 1, 5, 11, and 12 of Table 1 and Table 3 were analyzed using three-way factorial ANOVA with mSOD genotype (genotype), presence or absence of the CAT construct (cattype), and group (Co vs. heat pulse) as the three cross-classified main effects.

SOD overexpression and oxygen consumption:
Genotypes 1–5 of Table 4 are the same as genotypes 13, 14, 2, 4, and 9, respectively, of Table 1 and Table 3. Recall that there are 12 replicate vials for each genotype assayed for oxygen consumption at multiple time points (dates), and 6 vials were control and 6 vials were heat pulsed. First, the two-factor fixed-effects ANOVA model described above was used to confirm that heat pulse caused increased life span in this experiment, and results are presented in the text.

Second, oxygen consumption was analyzed across all five genotypes (Table 4) using a three-way factorial ANOVA with main effects of genotype, date of assay, and group (Co vs. heat pulse), and results are presented in the text.

Third, the relationship between life span and oxygen consumption was analyzed across all five genotypes (Table 4) using ANCOVA of the line means, and results are presented in the text. The analysis was done in three steps: (i) correlation analysis of the means for oxygen consumption and life span; (ii) a model of "oxygen consumption = genotype + group"; and (iii) life span was included as a covariate in a model of "oxygen consumption = life span + genotype + group."

The more appropriate analysis for the conditional system was to analyze the relationship between the change in life span and the change in oxygen consumption caused by the heat pulse. An ANCOVA model was used that combined a continuous independent variable [percentage change in oxygen consumption (oxyper)] with a categorical variable (genotype) as

where Y_i is the percentage change in life span based on means (heat pulse - Co), µ_.. is an overall mean term, ß_i is the genotype effect, is the coefficient for X_i (is it significant?), and X_i is the percentage change in oxygen consumption. In this case genotype had two values: experimental (genotypes 1–4 of Table 4) and control (genotype 5 of Table 4). Results are presented in the text.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

FLP-out:
In the FLP-out approach, the yeast FLP recombinase is expressed under the control of the hsp70 heat-inducible promoter in one transgenic construct, called FLP. A brief heat stress causes tissue-general expression of FLP recombinase. A second transgenic construct (the "target" construct) contains the gene of interest downstream of the constitutive, tissue-general Drosophila actin5C promoter. Transcripts initiating at the actin5C promoter are prevented from reaching the gene of interest by a transcriptional "stop" sequence. This transcriptional stop sequence is itself flanked by FRTs, which are the target sites for FLP recombinase. After FLP expression is induced by the heat pulse, the FLP recombinase protein causes the precise excision of the transcriptional stop sequence in the target construct. This results in direct fusion of the actin5C promoter to the gene of interest and thus constitutive expression of the gene of interest from that point in time onward in all tissues of the fly. The particular site of chromosomal insertion affects the expression level of transgenes, and it was anticipated that MnSOD transgene expression levels would vary between lines. For this reason, six independent transgenic lines were generated, each containing the mSOD target construct integrated at one unique location in the genome.

Induced expression of MnSOD transgene increases MnSOD enzyme activity:
MnSOD enzyme activity was assayed in extracts of flies containing both FLP3 and the mSOD target construct. Induced overexpression of MnSOD enzyme varied from 29 to 75%, depending on the particular transgenic line (Table 1, genotypes 1–6). The MnSOD enzyme activity assay was somewhat variable, and for three of the six MnSOD overexpressing lines (genotypes 2, 3, and 6) the increase in enzyme activity had a P value slightly >0.05 by two-sided t-test. However, when considered as a group the MnSOD overexpressing lines had a highly significant increase in enzyme (average 45%) that was not observed in the control lines, as confirmed by two-factor fixed-effects ANOVA (Table 2).

Genotype 1 differs from the others in that the insert is on the X chromosome. Repeat of the ANOVA analysis of enzyme activity excluding genotype 1 yields essentially the same results: group F = 57.00, P < 0.0001; genotype F = 10.52, P < 0.0001; group x genotype F = 0.81, P = 0.5343.

Heat pulse alone does not increase MnSOD enzyme activity:
The control lines (Table 1, genotypes 7–9) do not contain a MnSOD transgene, and for these lines there was an average 14% increase in MnSOD enzyme activity that was not significant. Two-factor fixed-effects ANOVA results were group F = 4.34, P = 0.0592; genotype F = 147.41, P < 0.0001; group x genotype F = 0.43, P = 0.5587.

Induced expression of MnSOD transgene increases mean and maximum life span:
Mean life span was assayed in control and experimental (HP) populations of 300 flies each, for each MnSOD transgenic line (Table 3, genotypes 1–6). Mean life span was increased by an average of 16%, with some lines showing 30–33% increases. Maximum life span was assayed by determining the time until 90% mortality. Maximum life span was increased by an average of 15%, with one line showing as much as 37% increase. Analysis of all the MnSOD transgenic lines using ANOVA confirmed that both mean and maximum life span were significantly increased.

Two-factor fixed-effects ANOVA results for mean life span were group F = 353.80, P < 0.0001; genotype F = 531.96, P < 0.0001; group x genotype F = 18.93, P < 0.0001. Because the group x genotype interaction was significant, it may be informative to repeat the ANOVA separately for group = control and group = HP. When this was done the genotype effect was unchanged in each case (P < 0.0001).

Repeat of the ANOVA analysis of mean life span excluding genotype 1 yielded essentially the same results: group F = 261.63, P < 0.0001; genotype F = 363.60, P < 0.0001; group x genotype F = 21.78, P < 0.0001. Because the group x genotype interaction was significant, it may be informative to repeat the ANOVA separately for group = control and group = HP. When this was done the genotype effect was unchanged in each case (P < 0.0001).

Maximum life span data for the MnSOD overexpressing genotypes 1–6 (Table 3) were analyzed using the same two-factor fixed-effects ANOVA model. Results were group F = 20.97, P = 0.0006; genotype F = 34.67, P < 0.0001; group x genotype F = 1.77, P = 0.1943.

Repeat of the analysis of maximum life span excluding genotype 1 yielded essentially the same results: group F = 16.49, P = 0.0023; genotype F = 46.50, P < 0.0001; group x genotype F = 1.54, P = 0.2629.

As expected, the transgenic lines varied in their starting life spans, due to unavoidable small differences in genetic background (KAISER et al. 1997 ; SUN and TOWER 1999 ; TATAR 1999 ; TOWER 1996 , TOWER 2000 ). The largest increase in mean life span (avg. 33%) was observed for line mSODX1 (Table 3, genotype 1), which has the lowest starting life span of 26 days. The possibility cannot be ruled out that, for this particular strain, MnSOD overexpression is rescuing some unique genetic defect. However, the starting life spans of genotypes 2–4 range from 40 to 56 days, which is typical for Drosophila controls, and these strains exhibit reproducible 12–29% increases in life span. In addition, ANOVA analysis of the MnSOD transgenic lines, omitting line mSOD(X)1, yielded qualitatively identical results. Therefore the data demonstrate that MnSOD overexpression extends life span regardless of the starting life span of the strain. Similar results were obtained in the previous study of Cu/ZnSOD overexpression; i.e., life span was extended in both long- and short-lived genetic backgrounds with the largest increases observed in the short-lived background (SUN and TOWER 1999 ).

Heat pulse alone does not increase life span:
The control strains (Table 3, genotypes 7–9) that do not contain a MnSOD transgene did not exhibit a significant increase in mean or maximum life span due to the heat pulse, as confirmed by two-factor fixed-effects ANOVA. Results for mean life span were group F = 1.02, P = 0.3125; genotype F = 37.06, P < 0.0001; group x genotype F = 2.91, P = 0.0544. Results for maximum life span were group F = 0.02, P = 0.8911; genotype F = 8.59, P = 0.0173; group x genotype F = 4.67, P = 0.0598. Therefore the heat pulse alone does not increase life span, and increased life span must result from MnSOD overexpression. Representative survival curves are presented for three MnSOD overexpressing lines (Fig 1, A–C) and three control lines (Fig 1, D–F).

Life span is increased in proportion to the increase in MnSOD enzyme activity:
Inspection of Table 1 and Table 3 suggests that the largest increases in life span are observed in the lines yielding the largest increases in enzyme activity. Each transgenic line varies in its starting enzyme activity and life span. Plotting the percentage change in life span vs. the percentage change in enzyme activity corrects for this variation. The plot reveals a strikingly linear correlation between enzyme activity and both mean and maximum life span for all six MnSOD transgenic lines (Fig 2). Regression analysis demonstrates that this relationship is significant for both enzyme activity and mean life span and enzyme activity and maximum life span. Results for mean life span were R² = 0.9375, the intercept term was significant P = 0.0184, and the slope term was significant P = 0.0015. Results for maximum life span were R² = 0.8283, the intercept term was not significant P = 0.0790, and the slope term was significant P = 0.0118.

Catalase overexpression is not associated with increased life span:
FLP-out using the FLP3 insertion allowed induced overexpression of catalase up to 37%, as measured by enzyme assay of fly extracts (Table 1). Increased catalase activity has previously been shown to increase resistance to killing by H₂O₂ feeding, but to have neutral or slightly negative effects on life span on its own or when coexpressed with Cu/ZnSOD (SUN and TOWER 1999 ). In the experiments presented here, catalase was again found to have neutral or negative effects on life span on its own (Table 3, genotype 10) and to have neutral or negative effects on life span when coexpressed with MnSOD (Table 3, compare genotypes 1 and 5 with genotypes 10 and 11). The neutral or slightly negative effects of catalase when combined with MnSOD may be due in part to the fact that MnSOD overexpression was significantly reduced when the catalase construct was also present. A similar effect was previously observed for simultaneous overexpression of catalase and Cu/ZnSOD (SUN and TOWER 1999 ). Taken together, the data support the conclusion that catalase activity is already in excess in the adult fly, at least with regard to life span.

The results were confirmed by three-factor fixed-effects ANOVA. Results for MnSOD enzyme activity were cattype F = 114.00, P < 0.0001; genotype F = 67.47, P < 0.0001; cattype x genotype F = 10.65, P = 0.0049; group F = 58.06, P < 0.0001; cattype x group F = 7.52, P = 0.0145; genotype x group F = 0.52, P = 0.4801; cattype x genotype x group F = 2.15, P = 0.1624.

Results for mean life span were cattype F = 4.89, P = 0.0271; genotype F = 452.39, P < 0.0001; cattype x genotype F = 22.11, P < 0.0001; group F = 87.18, P < 0.0001; cattype x group F = 34.71, P < 0.0001; genotype x group F = 11.74, P = 0.0006; cattype x genotype x group F = 0.33, P = 0.5665.

Results for maximum life span were cattype F = 0.00, P = 0.9610; genotype F = 9.47, P = 0.0152; cattype x genotype F = 0.74, P = 0.4161; group F = 8.86, P = 0.0177; cattype x group F = 1.59, P = 0.2428; genotype x group F = 0.43, P = 0.5304; cattype x genotype x group F = 1.35, P = 0.2794.

SOD overexpression and oxygen consumption:
The effect of SOD overexpression on metabolic rate of the flies was examined by assaying O₂ consumption at time points throughout the adult life span. In addition to the MnSOD lines, two Cu/ZnSOD lines were also examined, and reassay of enzyme activity and life span for the Cu/ZnSOD lines yielded results essentially identical to those previously reported (SUN and TOWER 1999 ).

ANOVA (MATERIALS AND METHODS) was first used to confirm that life span was significantly increased in both the Cu/ZnSOD and MnSOD overexpressing lines (Table 4, genotypes 1–4) but was not increased in the Oregon-R controls (genotype 5). When the two Cu/ZnSOD-containing genotypes (Table 4, genotypes 1 and 2) were analyzed together for mean life span the increase was significant: genotype F = 66.79, P < 0.0001; group F = 31.19, P < 0.0001; genotype x group F = 1.47, P = 0.2257. When the two MnSOD-containing genotypes (Table 4, genotypes 3 and 4) were analyzed together for mean life span the increase was significant: genotype F = 140.28, P < 0.0001; group F = 71.39, P < 0.0001; genotype x group F = 2.11, P = 0.1465. When all four SOD-containing genotypes (Table 4, genotypes 1–4) were analyzed together the increase was significant: genotype F = 536.17, P < 0.0001; group F = 102.63, P < 0.0001; genotype x group F = 7.16, P < 0.0001.

Second, oxygen consumption was analyzed across all five genotypes (Table 4) using a three-way factorial ANOVA with main effects of genotype, date of assay, and group (Co vs. heat pulse). Results for oxygen consumption were genotype F = 7.16, P < 0.0001; group F = 12.54, P = 0.0005; genotype x group F = 0.94, P = 0.4433; date F = 3.61, P < 0.0001; genotype x date F = 0.73, P = 0.7242; group x date F = 0.53, P = 0.9131; genotype x group x date F = 0.37, P = 0.9727.

Third, the relationship between life span and oxygen consumption was analyzed across all five genotypes (Table 4) using ANCOVA of the line means. The analysis was done in three steps.

1. Correlation analysis of the means for oxygen consumption and life span revealed a negative correlation: -0.75497.

2. A model of "oxygen consumption = genotype + group" gave results genotype F = 26.84, P = 0.0038; group F = 21.04, P = 0.0101.

3. Life span was included as a covariate in a model of "oxygen consumption = life span + genotype + group" with results life span F = 0.05, P = 0.8322; genotype F = 8.47, P = 0.0552; group F = 8.82, P = 0.0591.

The results revealed that heat pulse had a significant negative effect on oxygen consumption across all five genotypes and that there was a negative correlation between life span and oxygen consumption. However, the analyses did not control for the different starting life spans and oxygen consumption values of the various genotypes. These base line life spans and oxygen consumption values are expected to correlate strongly and may mask the effects caused by transgene expression. The more appropriate analysis for the conditional system was therefore to analyze the relationship between the change in life span and the change in oxygen consumption caused by the heat pulse. An ANCOVA model was used that combined a continuous independent variable (oxyper) with a categorical variable (genotype) as described in MATERIALS AND METHODS. In this case genotype had two values: experimental (genotypes 1–4 of Table 4) and control (genotype 5 of Table 4). Results were genotype F = 11.36, P = 0.0434; oxyper F = 0.70, P = 0.4651. Consistent with the analyses presented above, there was a genotype effect, meaning the experimental genotypes lived longer than control. In contrast, the coefficient for oxygen was not significant, meaning oxygen consumption was not a significant factor in increasing life span in experimentals relative to control. Therefore, increased life span was not associated with decreased O₂ consumption as is found when life span is altered by culture temperature (MIQUEL et al. 1976 ). The data support the conclusion that overexpression of MnSOD or Cu/ZnSOD increases life span by a mechanism that does not involve reductions in oxidative metabolism.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To further test the theory that oxidative stress is a major cause of aging, Drosophila were engineered to overexpress the antioxidant enzyme MnSOD. The FLP-out transgenic system yields transgene expression specifically in the adult in all tissues. The amount of MnSOD enzyme overexpression achieved varied between six independent transgenic lines, with increases up to 75%. Life span was found to be increased in proportion to the increase in enzyme. Mean life span was increased up to 33%, and maximum life span, as measured by time to 90% mortality, was increased up to 37%. Therefore, adult Drosophila life span is limited by MnSOD activity, analogous to the results previously obtained for Cu/ZnSOD (PARKES et al. 1998 ; SUN and TOWER 1999 ). Both Cu/ZnSOD and MnSOD were found to increase mean and maximum life span with no detectable negative effect on metabolic activity. Co-overexpression of catalase with MnSOD had no added benefit for life span, consistent with the previous conclusion that catalase is in excess in the adult fly with regard to life span (SUN and TOWER 1999 ).

Interestingly, it has recently been reported that flies transgenic for an extra copy of the native MnSOD gene did not exhibit increased life span (MOCKETT et al. 1999 ). This difference in results is not due simply to a difference in the amount of enzyme overexpression achieved, as the levels of enzyme overexpression observed in whole fly extracts were similar to the levels of overexpression observed here. We suggest two possible models for the difference in results obtained with the native promoter approach vs. the FLP-out approach. In the first model, MnSOD overexpression during development is hypothesized to be deleterious and have negative effects on subsequent adult life span. The native promoter approach is expected to result in overexpression throughout the life cycle both during development and in the adult. Negative effects of MnSOD overexpression during development might then cancel out any positive effects on life span that might have otherwise resulted from overexpression in the adult. In contrast, with FLP-out MnSOD overexpression is specific to the adult stage and therefore significant increases in life span are observed. However, this model requires that the putative positive and negative effects of the native promoter constructs be almost exactly equal to yield unchanged life span for both MnSOD and Cu/ZnSOD (as described below)—a result that seems unlikely. The second model, which we therefore favor, involves possible differences in the tissue specificity of expression. With the native promoter approach the tissue and temporal specificity of overexpression is expected to be the same as, or similar to, the native expression pattern. In contrast, with FLP-out MnSOD overexpression is ultimately driven by the powerful, tissue-general actin5C promoter. We hypothesize that the actin5C promoter results in a novel pattern of MnSOD transgene expression that results in increased life span. The actin5C promoter may drive MnSOD expression in some critical cells or tissues where the native promoter is inactive or less active. This situation appears to be analogous to that with Cu/ZnSOD. Overexpression of Cu/ZnSOD alone with the native promoter approach does not yield increased life span, as previously reported (ORR and SOHAL 1993 ) and in extensive subsequent analyses of the same and additional transgenic lines (B. ORR, personal communication). In contrast, overexpression of Cu/ZnSOD with the heterologous FLP-out system or the heterologous Gal4/UAS system does yield increased life span (PARKES et al. 1998 ; SUN and TOWER 1999 ). In the latter case life span increase is associated with preferential expression of Cu/ZnSOD in the motorneurons. Therefore, for both Cu/ZnSOD and MnSOD overexpression using the native promoter does not yield increased life span while the heterologous expression systems do yield increased life span. It will be of great interest in the future to determine what are the unique aspect(s) of the heterologous expression systems that allow for increased life span.

The present data for MnSOD and Cu/ZnSOD combined with previous studies of Cu/ZnSOD (PARKES et al. 1998 ; SUN and TOWER 1999 ) demonstrate that SOD activity is limiting for life span in the adult fly. Because the primary catalytic activity of SOD is to convert superoxide to H₂O₂, the current data suggest that increased life span results from more efficient conversion of superoxide and reduced superoxide levels. The first mechanism by which superoxide levels might limit life span is by directly or indirectly causing oxidative damage to cellular macromolecules and organelles. However, alternative mechanisms involving a negative signaling or regulatory effect of superoxide on life span or a positive signaling or regulatory effect of H₂O₂ on life span cannot be ruled out at this time. It will be of interest in the future to determine if overexpression of SOD and increased life span correlate with decreased and/or delayed accumulation of specific oxidative damage products as predicted by the first proposed mechanism. Identification of specific cellular molecules that are protected from oxidative damage by both MnSOD and Cu/ZnSOD overexpression may reveal critical life span-limiting targets for oxidative damage during aging. Of particular interest may be mitochondrial aconitase and the inner mitochondrial membrane protein adenine nucleotide translocase (ANT). Both proteins appear to be preferentially susceptible to oxidative damage during aging in houseflies (YAN et al. 1997 ; YAN and SOHAL 1998 ).

The results presented here are consistent with previous and concurrent studies of genetically selected Drosophila strains. Selection of genetically heterogeneous populations of Drosophila for late life female fecundity over many generations results in populations with correlated phenotypes of increased life span and increased stress resistance (LUCKINBILL et al. 1984 ; ROSE 1984 ; PARTRIDGE and FOWLER 1992 ). Detailed analysis of one such selected strain and its matched control strain reveals that increased life span in this particular strain correlates with increased oxidative stress resistance and increased expression of antioxidant enzyme genes, including Cu/ZnSOD and MnSOD (ARKING et al. 1991 , ARKING et al. 2000 ). The results presented here are also consistent with genetic selection experiments with regard to metabolic activity. Metabolic activity was assayed in the selected and control population as well as an inbred long-lived strain and inbred control strain derived from the selected and control populations. The results indicated that while aerobic efficiency declined as a function of age in all flies, metabolic activity of the long-lived and control strains was equal throughout the majority of the life span (ARKING et al. 1988 ; ROSS 2000 ; MOCKETT et al. 2001 ). Therefore both genetic selection experiments and FLP-out overexpression of SOD appear to increase life span by a mechanism that does not involve decreased metabolic activity.

It will be of interest to determine if SOD overexpression can extend life span in mammals. Consitutive overexpression of Cu/ZnSOD has been achieved in mice; however, no increases in life span have been observed (HUANG et al. 2000 ). Analogous to the situation with Drosophila, it may be found that inducible or tissue-specific expression systems (LI et al. 1999 ) will be required to observe increased life span. Finally, in Caenorhabditis elegans and Drosophila, and possibly also in yeast and mammals, a conserved insulin-like signal transduction pathway negatively regulates life span (KENYON 2001 ). In C. elegans the increased life span is associated with increased expression of the gene encoding mitochondrial MnSOD (HONDA and HONDA 1999 ). The data presented here demonstrate that in Drosophila MnSOD overexpression is sufficient to significantly increase life span. These results suggest the possibility that increased MnSOD expression may be responsible for all or part of the life span increases observed upon mutation of the conserved insulin-like life span regulatory pathway in C. elegans, Drosophila, and perhaps other species.

	ACKNOWLEDGMENTS

We thank Bill Orr and Raj Sohal for helpful comments on the manuscript and for communicating unpublished results, and we thank John Molitor for expert assistance with statistical analyses. This work was supported by grants from the Department of Health and Human Services, National Institute on Aging to J.T. (AG11644) and from the National Science Foundation to T.B. (IBN9723404).

Manuscript received September 28, 2000; Accepted for publication February 14, 2002.

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				W. C. Orr, R. J. Mockett, J. J. Benes, and R. S. Sohal Effects of Overexpression of Copper-Zinc and Manganese Superoxide Dismutases, Catalase, and Thioredoxin Reductase Genes on Longevity in Drosophila melanogaster J. Biol. Chem., July 11, 2003; 278(29): 26418 - 26422. [Abstract] [Full Text] [PDF]

				D. Pratic̣ Lipid Peroxidation and the Aging Process Sci. Aging Knowl. Environ., December 18, 2002; 2002(50): re5 - 5. [Abstract] [Full Text] [PDF]

				K. Kirby, J. Hu, A. J. Hilliker, and J. P. Phillips RNA interference-mediated silencing of Sod2 in Drosophila leads to early adult-onset mortality and elevated endogenous oxidative stress PNAS, December 10, 2002; 99(25): 16162 - 16167. [Abstract] [Full Text] [PDF]

				A. Dillin, D. K. Crawford, and C. Kenyon Timing Requirements for Insulin/IGF-1 Signaling in C. elegans Science, October 25, 2002; 298(5594): 830 - 834. [Abstract] [Full Text] [PDF]