Free Radical Biology & Medicine 47 (2009) 803–813

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Free Radical Biology & Medicine

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Original Contribution

Sod2 knockdown in the musculature has whole-organism consequencesin Drosophila

Ian Martin a, Melanie A. Jones a, Devin Rhodenizer a, Jie Zheng b, John M. Warrick c,Laurent Seroude b, Mike Grotewiel a,⁎a Department of Human and Molecular Genetics, Virginia Commonwealth University, Richmond, VA 23113, USAb Department of Biology, Queen's University, Kingston, Canada ON K7L 3N6c Biology Department, University of Richmond, Richmond, VA 23173, USA

⁎ Corresponding author.E-mail address: msgrotewiel@vcu.edu (M. Grotewie

0891-5849/$ – see front matter © 2009 Elsevier Inc. Adoi:10.1016/j.freeradbiomed.2009.06.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 25 March 2009Revised 12 June 2009Accepted 17 June 2009Available online 21 June 2009

Keywords:SOD2MuscleMitochondriaFree radicals

Oxidative damage to cell macromolecules by reactive oxygen species is associated with numerous diseasesand aging. In Drosophila, RNAi-mediated silencing of the mitochondrial antioxidant manganese superoxidedismutase (SOD2) throughout the body dramatically reduces life span, accelerates senescence of locomotorfunction, and enhances sensitivity to applied oxidative stress. Here, we show that Sod2 knockdown in themusculature alone is sufficient to cause the shortened life span and accelerated locomotor declines observedwith knockdown of Sod2 throughout the body, indicating that Sod2 deficiency in muscle is central to thesephenotypes. Knockdown of Sod2 in the muscle also increased caspase activity (a marker for apoptosis) andcaused a mitochondrial pathology characterized by swollen mitochondria, decreased mitochondrial content,and reduced ATP levels. These findings indicate that Sod2 plays a crucial role in the musculature in Drosophilaand that the consequences of SOD2 loss in this tissue extend to the viability of the organism as a whole.

© 2009 Elsevier Inc. All rights reserved.

The electron transport chain (ETC) in mitochondria is the primary

source of reactive oxygen species (ROS) in eukaryotes, with 0.1% ormore of oxygen entering the chain being univalently reduced tosuperoxide [1,2]. Superoxide escaping from the ETC and other ROSderived from superoxide represent a significant oxidative threat tomitochondria and cells as a whole [3]. Superoxide dismutase (SOD; EC1.15.1.1) enzymes are antioxidants that protect cells from oxidativestress by catalyzing the dismutation of superoxide to oxygen andhydrogen peroxide. Hydrogen peroxide is subsequently reduced towater by catalase (EC 1.11.1.6) or a number of peroxidases [4].

The fruit fly, Drosophila melanogaster, possesses the three majoreukaryotic SOD isoforms: SOD1, found predominantly in the cytosol;SOD2, in the mitochondrial matrix; and SOD3, in the extracellularspace [5]. The localization of SOD2 to the mitochondrial matrix placesit in close proximity to the ETC, suggesting that this enzyme has animportant role in protecting cells from superoxide produced bymitochondrial respiration. Accordingly, complete loss of SOD2 activityin mice results in a number of pathological conditions, includingdilated cardiomyopathy, accumulation of lipid in liver and skeletalmuscle, motor disturbances with neurodegeneration (in a mixedC57BL/6 and 129Sv background), and a very short life span [6–8]. Atthe cellular level, Sod2 mutant mice exhibit increased oxidativedamage to lipid in addition to genomic and mitochondrial DNA [9,10],decreased mitochondrial respiration rates, deficits in ETC activity

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reminiscent of mitochondrial disease [11], and elevated hepaticapoptosis [9]. In Drosophila, knockout or knockdown of Sod2throughout the body also severely shortens life span [12–15].Furthermore, Sod2 deficient flies exhibit accelerated declines inolfactory and locomotor behavior with age in addition to neurode-generation associated with elevated apoptosis [14,16]. All of thesestudies highlight the importance of SOD2 in protecting cells andtissues from oxidative damage.

In this study we investigated whether Sod2 functions withinspecific tissues to support normal life span and locomotor function.We found that expression of Sod2 RNAi transgenes in themusculature,but not the nervous system, dramatically shortens life span andaccelerates loss of locomotor behavior across age. Flies with knock-down of Sod2 inmuscle exhibit mitochondrial pathology, reduced ATPcontent, and elevated caspase activity, supporting the hypothesis thatmitochondrial dysfunction in the musculature is central to theshortened life span and accelerated decline in locomotor function inthese animals.

Materials and methods

Fly stocks and husbandry

Flies were reared to adulthood and housed for all aging studies at25°C and 55% relative humidity under a 12-h light–dark cycle on asugar:yeast:cornmeal:agar medium (10%:2%:3.3%:1% w/v) supple-mented with 0.2% Tegosept (Sigma Chemical Co., St. Louis, MO, USA)

Fig. 1. Ubiquitous expression of Sod2 RNAi reduces SOD2 activity, shortens life span, andaccelerates locomotor senescence. (A) Protein extracts (45 μg total protein/lane) fromadult males (∼2 days of age) were electrophoresed on native polyacrylamide gels. SODactivity was visualized using an in-gel assay as described under Materials and methods.(B) Mean and median life span in flies expressing Sod2-IR via da-Gal4 (da-Gal4; Sod2-IR24) were reduced compared to control flies harboring da-Gal4 or Sod2-IR24transgenes alone (log-rank test, pb0.0001, N=150 flies per genotype). (C) Age-relatedloss of negative geotaxis was accelerated in flies expressing Sod2-IR via da-Gal4 (da-Gal4; Sod2-IR24) compared to da-Gal4 and Sod2-IR24 control flies (two-way ANOVA,pb0.0001, N=5 vials of 25 adult males per genotype). Negative geotaxis data aremeans±SEM.

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and active yeast. Sod2 was knocked down by using the Gal4/UASsystem [17] to express Sod2 inverted-repeat (Sod2-IR) transgenespreviously described [13]. The UAS-Sod2-IR24, UAS-Sod2-IR15, anddaughterless-Gal4 (da-Gal4) lines were provided by John Phillips(University of Guelph, ON, Canada). Other fly lines were from thefollowing sources: Mef2-Gal4, Sunita Gupta Kramer (Rutgers); Appl-Gal4; Lawrence Goldstein (University of California at San Diego, LaJolla, CA, USA); D42-Gal4, Gabrielle Bouilanne (University of Toronto,ON, Canada); GMH5-Gal4, R. J. Wessells (University of Michigan, AnnArbor, USA); 24B-Gal4, repo-Gal4, and elav-Gal4, Drosophila StockCenter (Bloomington, IN, USA); 91Y-Gal4 and 188Y-Gal4, J. DouglasArmstrong (University of Edinburgh, UK); and UAS-Shibirets1, RonDavis (Baylor College of Medicine, Waco, TX, USA). Flies with a secondchromosome harboring the UAS-lacZ and UAS-Sod2-IR24 transgeneswere generated by recombination and confirmed by PCR using theprimers 5′-GAACACGTCGCTAAGCGAAAGC-3′ and 5′-GCGAAATTT-TACGGGCCACGAAC-3′ to detect UAS-Sod2-IR and the primers 5′-CTGACGGGCTCCAGGAGTC-3′ and 5′-CTACCGGATTGATGGTAGTGGTC-3′ to detect UAS-lacZ.

SOD activity

Groups of 25 adult males (0–3 days of age) per genotype werecollected under brief CO2 anesthesia and homogenized in extractionbuffer (50 mM potassium phosphate, 0.1 mM EDTA, 2% Triton X-100,pH 7.8) on ice. To rupture mitochondria, samples were sonicated for20 s and then incubated at 4°C for 45 min. Ruptured mitochondrialsamples were centrifuged at 16,000 g for 15 min at 4°C and theresulting supernatant was stored at 4°C. Protein concentration wasdetermined using the Lowry method (DC protein assay; Bio-Rad,Hercules, CA, USA). Samples containing equal amounts of proteinwereelectrophoresed by discontinuous native PAGE (4% stacking gel (pH6.8), 20% resolving gel (pH 8.8)) in sample buffer (0.5 M Tris–HCl, 50%glycerol, 0.01% bromophenol blue) at 80–100 V. SOD activity wasmeasured colorimetrically using a modified version of an in-gel assaypreviously described [13]. Briefly, gels containing electrophoresedprotein samples were incubated in 2.5 mM nitroblue tetrazolium and50 mM potassium phosphate buffer for 20 min in the dark undergentle agitation. Gels were washed briefly in 50 mM phosphate bufferand then incubated in 28 mM N,N,N′,N′-tetramethylethylenediamine,28 μM riboflavin, 50 mM potassium phosphate for 15 min in the darkwith gentle agitation. After a brief wash in 50 mM potassiumphosphate buffer, gels were exposed to white light for ∼15 min forfull color development. In-gel SOD activity was quantified bydensitometry using Alpha Imager software (Alpha Innotech Corp.,San Leandro, CA, USA).

Life span analysis

Adult males (1–3 days of age, 100–200 flies per genotype) werecollected under brief CO2 anesthesia and transferred to fresh food vialsat a density of 25 flies per vial. Flies were transferred to fresh food vialsevery 2–4 days. Dead and surviving flies were counted during eachtransfer to fresh food.

Negative geotaxis

Groups of 25male flies (1–3 days of age)were collected under briefCO2 anesthesia and allowed to recover for at least 18 h at 25°C and 55%relative humidity before assay. Negative geotaxis (bang-inducedclimbing) was measured in rapid iterative negative geotaxis (RING)assays as previously described [18]. Flies were transferred to a RINGapparatus and rested for 1 min. Negative geotaxis was initiated bysharply rapping the apparatus on a table three times in rapidsuccession. The flies' positions in the tubes were captured in digitalimages taken 4 s after initiating the behavior. Data (distance climbed

for each fly) were extracted from the images using Scion Image asdescribed [18]. The performance of flies in a single vial in fiveconsecutive trialswas averaged to generate a single datum. Five vials offlies per genotype were tested to generate N=5 for each genotype inindividual experiments. After each RING test, flies were transferred tofresh food vials and housed until the next assessment of negativegeotaxis.

Determination of decline-time 50 (DT50; time required fornegative geotaxis to decline to 50% of the youngest flies) wasperformed as described previously [19]. DT50 values, interpolatedfrom second-order curve fits using Prism 4.02 (GraphPad Software,San Diego, CA, USA), were determined for each vial of flies individuallyand then compiled for each genotype.

Temperature-sensitive Shibire (Shits1) was expressed using theGal4/UAS system [17]. Shits1 encodes a dominant-negative dynaminthat blocks neurotransmission at chemical synapses at 31°C [20,21].Adult flies expressing Shits1 (containing a Gal4 driver and two UAS-Shits1 transgenes) and control flies (containing the UAS-Shits1

transgenes but no Gal4 driver) were collected and allowed to recover

Fig. 2. Gal4 drivers for neurons involved in negative geotaxis. There was a significantoverall effect of genotype (i.e., Gal4 driver used) on behavior (one-way ANOVA,pb0.0001, N=4 or 5 (representing 100–125 flies) for Gal4;Shits1 flies; N=35(representing 875 flies) for Shits1/+ control). (A) Expression of Shits1 via the Gal4drivers 91Y, 188Y, Appl, D42, and OK307 caused a temperature-sensitive disruption ofnegative geotaxis (Tukey HSD multiple comparison, pb0.05). (B) Negative geotaxiswas not significantly affected by temperature in flies expressing Shits1 via all otherGal4 drivers (Tukey HSD multiple comparison). Data (means±SEM, compiled fromseven experiments) are the percentage negative geotaxis observed at 25°C remainingat 31°C.

Fig. 3. Nervous system Sod2-IR expression has modest effects on locomotorsenescence and life span. Sod2-IR expression via the panneuronal drivers (A) 188Y-Gal4, (B) 91Y-Gal4, (C) Appl-Gal4, or (D) elav-Gal4 significantly impaired negativegeotaxis behavior across age (individual two-way ANOVAs for effect of age andgenotype, pb0.0001 for both factors, N=5–10 vials of 25 males per genotype).Tukey's HSD posttest revealed that all Sod2 knockdown lines performed significantlyworse across age than either of their control groups (pb0.05). Negative geotaxis data(mean±SEM) are compiled from two independent experiments except in (D), whichis from one experiment. Life span was also reduced in flies (150 per genotype) afterSod2-IR expression via (E) 188Y-Gal4 or (F) elav-Gal4. See Table 1 for percentagedecrease in median life span.

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as above. Negative geotaxis was first assessed in RING assaysperformed at 25°C. Flies were allowed to recover for 1 h, transferredto 31°C for 30 min, and then tested again in RING assays at theelevated (i.e., restrictive) temperature. The effect of Shits1 expressionon RINGwas quantitated as percentage negative geotaxis remaining atthe restrictive temperature ((negative geotaxis at 31°C)/(negativegeotaxis at 25°C) × 100%) for each genotype.

Expression patterns of Gal4 drivers and β-galactosidase histology

Gal4 expression was assessed by using a UAS-lacZ reporter gene ina Sod2 knockdown background. Flies with a double-transgenic UAS-lacZ, UAS-Sod2-IR24 second chromosome (generated by recombina-tion) were mated to Gal4 driver stocks to produce progeny withconcurrent expression of lacZ and knockdown of Sod2.

The spatial expression patterns of da-Gal4, Mef2-Gal4, and 24B-Gal4 across age were assessed by β-galactosidase histology. Briefly,adult flies were collected under brief CO2 anesthesia, embedded inTissue-Tek OCT (Sakura Finetak USA, Inc.), transferred in embeddingmedium to a specimen holder, and frozen at −40°C. Whole-bodytissue sections (15 μm) were cut using a Hacker Bright cryostat(Model OTC5000; GMI, Ramsey, MN, USA) at −20°C. Sections were

transferred to slides, allowed to air dry for 1 h at room temperature,and then fixed in 2% glutaraldehyde for 20 min. Slides were washedtwice for 5 min in phosphate-buffered saline (PBS) and placed at 37°Cuntil dry. β-Galactosidase (EC 3.2.1.23) activity was visualized byadding prewarmed X-gal substrate to the slides for 30min. Slideswerewashed twice for 5 min in PBS and coverslips mounted in 70% glycerolin PBS. Digital images were obtained using a Zeiss Axioplan-2microscope, Axiocam CCD camera, and Axiovision software (CarlZeiss, Oberkochen, Germany).

Quantitative assessment of whole-body lacZ expression across agewas performed by spectrophotometric assessment of β-galactosidaseactivity as previously described [22]. Three adult males werehomogenized in extraction buffer (50 mM potassium phosphate,1 mM MgCl2, 0.5 μg/ml leupeptin, 0.5 μg/ml aprotinin, 0.7 μg/mlpepstatin A, pH 7.2) at the indicated ages. Homogenates werecentrifuged at16,000g for 5min.β-Galactosidase activitymeasurements

Table 1Accelerated loss of negative geotaxis and shortened life span in flies with Gal4-drivenexpression of Sod2-IR

Gal4 driver Tissue specificity % ↓ in negativegeotaxis DT50

% ↓ in medianlife span

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(i.e., change in absorbance at 562 nm over 5 min in a Ultrospec 2000spectrophotometer (Pharmacia Biotech, Piscataway, NJ, USA)) in theresulting supernatants were initiated by addition of 100 μM chloro-phenol red-β-d-galactopyranoside. β-Galactosidase activity wasexpressed as the absorbance change per minute per milligram of homo-

da-Gal4 Ubiquitous 89 79Actin-Gal4 Ubiquitous nd 88elav-Gal4 Panneuronal 5 23188Y-Gal4 Panneuronal 2 1991Y-Gal4 Panneuronal 19 ndAppl-Gal4 Panneuronal 14 ndD42-Gal4 Motor neurons 17 5TH-Gal4 Dopaminergic neurons 15 ndc17-Gal4 Giant fiber system 0 12OK307-Gal4 Giant fiber system 0 19repo-Gal4 Panglial 14 22Mef2-Gal4 Panmuscle 94 9324B-Gal4 Panmuscle 89 95GMH5-Gal4 Cardiac muscle 26 11

The negative geotaxis decline time50 (DT50) is the time required for negative geotaxisperformance to decline by 50% of original values. Percentage decrease in DT50 ormedianlife span for each Sod2 knockdown line was calculated compared to the poorestperforming control containing Gal4 or Sod2-IR24 alone. nd, not determined.

genate protein. β-Galactosidase activity in control flies containing eitherthe UAS or the Gal4 transgenes alone was negligible.

Transmission electron microscopy

Thoraces were dissected from adult male flies under anesthesiaand immediately fixed in 1% glutaraldehyde, 2% paraformaldehyde,0.2 M sodium cacodylate (pH 7.4), osmicated (1% osmium tetroxide),stained (1% uranyl acetate), and dehydrated in an ethanol series withmicrowave assistance. Fixed and dehydrated samples were brieflyincubated in propylene oxide and then embedded in propylene oxide/Embed 812 resin. Thin sections (80–100 nm) were transferred tocopper grids and visualized using a JEOL 1010 transmission electronmicroscope (JEOL USA, Peabody, MA, USA). Mitochondrial content inrepresentative images was quantitated as the fraction of total imagearea occupied by mitochondria. Four to eight representative low-magnification images were analyzed for each genotype at each ageusing Scion Image software resulting in a total image field of 0.65–1.3 mm2 analyzed.

ATP content

ATP content was assessed using the ATP Bioluminescent Assay Kit(Sigma Chemical Co.). For each sample, five thoraces were dissectedfrom adult males under CO2 anesthesia, homogenized in 6 M guaninehydrochloride, and heated at 95°C for 5 min to eliminate endogenousATPase activity. Homogenates were centrifuged at 16,000 g for 15 minat room temperature and the resulting supernatantwas diluted 10-foldin sample buffer (0.2 M glycine, 50 mM MgCl2, 4 mM EDTA, pH 7.4).Luminescence was measured after addition of the ATP assay kitreagents according to themanufacturer's instructions in aWallac 1420Victor V plate reader (Perkin–Elmer, Waltham, MA, USA). ATP concen-

Fig. 4. Neuronal Sod2 knockdown impairs locomotor function across age and shortenssurvival. Sod2-IR24was expressed specifically (A and B) inmotor neurons via D42-Gal4,(C and D) in giant fiber neurons via OK307-Gal4 or (E and F) c17-Gal4, (G) indopaminergic neurons via TH-Gal4, and (H and I) throughout glia via repo-Gal4.Negative geotaxis performance across age (A, C, E, G, I) was significantly worse in fliesexpressing Sod2-IR24 via all Gal4 drivers except OK307-Gal4 in (C) (individual two-wayANOVAs for effect of age and genotype, pb0.0001, N=5–10 vials of 25 male flies forboth factors in all data sets, followed by Tukey's HSD posttest (pb0.05, or ns for OK307-Gal4;Sod2-IR24). Life span (B, D, F, and I) was significantly reduced in all Sod2knockdown genotypes (log-rank test p≤0.0263, 150 flies per genotype). Survival wasnot assessed for Sod2-IR24 expression via TH-Gal4.

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trations of thorax homogenates were interpolated from standardcurves using ATP standards.

Caspase activity

Caspase activity was assessed as the hydrolysis of the peptidesubstrate Ac-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) essentially as described [23]. Briefly, two thoraceswere homogenized in lysis buffer (50 mM Hepes, pH 7.5, 100 mMNaCl, 1 mM EDTA, 0.1% Chaps, 10% sucrose, 5 mM DTT, 0.5% TritonX-100, 4% glycerol) and centrifuged for 5 min at 13,000 g at 4°C togenerate a supernatant. The supernatant was incubated with 25 mMAc-DEVD-AMC for 1 h at 27°C and fluorescence of the AMC productwas determined using a Spectra Max fluorescence microplate reader(Molecular Devices, Sunnyvale, CA, USA) with excitation andemission set at 360 and 460 nm, respectively. Protease-specific

Fig. 5. Expression of Sod2-IR in muscle reduces life span and accelerates age-related locomoexpressed via (A and B)Mef2-Gal4, (C and D) 24B-Gal4, or (E and F) GMH5-Gal4. (A, C, and E(log-rank test, pb0.0001, N=150 flies per genotype). (B, D, and F) Age-related locomotor im(two-way ANOVA; effect of age and genotype, pb0.0001; Tukey's HSD multiple comparindependent experiments.

caspase product was determined by subtracting the fluorescence ofa duplicate sample pretreated with the caspase inhibitor Ac-DEVD-CHO (2.5 mM) for 15 min for each sample.

Statistical analyses

Statistical treatment of data (one- and two-way ANOVA, Bonfer-roni's multiple comparison tests, Tukey's honestly significant differ-ence (HSD) multiple comparison tests, log-rank tests) was performedwith JMP 5.01 (SAS Institute, Cary, NC, USA) and Prism 4.02 (GraphPadSoftware).

Results

Knockdown of Sod2 in Drosophila via RNA interference (RNAi)using Gal4 to ubiquitously express a UAS–Sod2-inverted repeat

tor impairment. Two independent Sod2-IR transgenes, Sod2-IR15 and Sod2-IR24, were) Life spanwas significantly shortened by expression of Sod2-IR via all three Gal4 driverspairment was accelerated in flies with Sod2-IR expression driven by all three Gal4 linesison, pb0.05; N=5 vials of 25 flies per genotype). Data are representative of two

Fig. 6. Sod2-IR expression in the musculature knocks down SOD2 activity. (A) Whole-body SOD activity. SOD activity was measured in whole-body extracts using an in-gelassay described under Materials and methods. Each lane contained 45 μg total proteinfrom adult males (∼2 days of age). Flies had the 24B-Gal4, Mef2-Gal4, or no Gal4 driveralong with the Sod2-IR24 (24), Sod2-IR15 (15), or no Sod2-IR transgene (+). (B)Quantitation of whole-body SOD activity by densitometry. Whole-body SOD2 activitywas significantly reduced in flies with Sod2-IR24 and Sod2-IR15 expression driven by24B-Gal4 and Mef2-Gal4 compared to controls with the Gal4 or the Sod2-IR transgenesalone (individual one-way ANOVAs, p≤0.0126, N=3 groups of 25 flies per genotype,followed by Tukey's HSD posttest, ⁎pb0.05). (C) Quantitation of whole-body SOD1activity. Whole-body SOD1 activity was not affected by genotype (individual one-wayANOVAs, not significant, N=3 groups of 25 flies per genotype). Data in B and C aremeans±SEM.

808 I. Martin et al. / Free Radical Biology & Medicine 47 (2009) 803–813

(UAS-Sod2-IR) transgene substantially reduces SOD2 enzymaticactivity, shortens life span, and elicits hallmarks of mitochondrialdysfunction that are consistent with oxidative damage [13]. Weconfirmed that ubiquitous Gal4-driven expression of Sod2-IRknocked down SOD2 (but not SOD1) activity (Fig. 1A) anddecreased the life span of males (Fig. 1B) and females [15].Additionally, ubiquitous expression of Sod2-IR caused a rapid age-dependent loss of negative geotaxis (i.e., bang-induced climbing, alocomotor behavior [24]) across age in males (Fig. 1C) and females[15]. Interestingly, negative geotaxis in 1-day-old Sod2 knockdownand control flies was indistinguishable, indicating that newlyemerged flies with ubiquitous expression of Sod2-IR are notglobally sick. This finding is consistent with a previous reportshowing that ubiquitous Sod2 knockdown does not affect eclosionof adult flies, possibly because oxygen metabolism or ROS signalingis fundamentally different during development and adulthood [13].

Expression of Sod2-IR in the nervous system has modest effects onlocomotor function and life span

We reasoned that expression of Sod2-IR in the nervous systemmight accelerate age-related impairment in negative geotaxis andshorten life span. Tyrosine hydroxylase-Gal4 (TH-Gal4) expresses indopaminergic neurons required for negative geotaxis [25], making it auseful tool for exploring the consequences of Sod2-IR expression inregions of the nervous system relevant to the behavior. To identifyadditional Gal4 lines for neurons involved in negative geotaxis, weassessed negative geotaxis in flies with expression of Shits1 (atemperature-sensitive dominant-negative dynamin that blocks neu-rotransmission [20,26,27]) via a number of different Gal4 drivers. Flieswith panneural expression of Shits1 via 188Y-Gal4, 91Y-Gal4 [28], andAppl-Gal4 [29] were largely paralyzed and had little or no negativegeotaxis behavior remaining at the restrictive temperature (31°C),consistent with inhibition of neurotransmission throughout thenervous system (Fig. 2A). Expression of Shits1 by the spatially restrictedGal4 drivers D42 (motor neurons [30]) andOK307 (giantfiber neurons[31]) also caused substantial defects in negative geotaxis at therestrictive temperature (Fig. 2A). In contrast, control flies with theUAS-Shits1 transgene and no Gal4 driver (Shits1/+) or flies withexpression of Shits1 in mushroom body, olfactory, gustatory, centralcomplex, or eye neurons via a number of other Gal4 drivers had normalnegative geotaxis at the restrictive temperature (Fig. 2B). Together,these studies indicate that 91Y,188Y, Appl,D42, OK307, and TH expressGal4 in neurons required for negative geotaxis.

Expression of Sod2-IR via the panneuronal Gal4 drivers 188Y, 91Y,and Appl caused modest but statistically discernable impairments innegative geotaxis across age compared to control flies harboringeither the Gal4 or the Sod2-IR transgene alone (Figs. 3A–3C).Expression of Sod2-IR using another panneuronal Gal4 driver, elav-Gal4 [32], likewise caused a subtle but statistically significant defect innegative geotaxis with age (Fig. 3D). Expression of Sod2-IR via 188Y-Gal4 and elav-Gal4 also decreased life spanmodestly (Figs. 3E and 3F).Additionally, expression of Sod2-IR in motor (D42-Gal4), dopaminer-gic (TH-Gal4), or giant fiber (OK307-Gal4 and C17-Gal4 [31]) neuronsrequired for negative geotaxis or throughout the glia (repo-Gal4 [33])caused subtle impairments in negative geotaxis across age and/ormarginally reduced life span (Fig. 4). The detrimental effects ofnervous system Sod2-IR expression on negative geotaxis and life span,however, were substantially smaller than those observed afterubiquitous Sod2-IR expression (Table 1).

Expression of Sod2-IR in the musculature severely curtails life span andimpairs locomotor function across age

In stark contrast to the modest effects of Sod2-IR expression in thenervous system, the expression of two independent Sod2-IR trans-

genes (Sod2-IR15 and Sod2-IR24) in the musculature using Mef2-Gal4[34] dramatically reduced life span (Fig. 5A) and greatly acceleratedthe age-related impairment in negative geotaxis (Fig. 5B) compared tocontrol flies with either the Gal4 driver alone or the Sod2-IR transgenealone. Sod2-IR expression in the musculature via 24B-Gal4 [35] alsoshortened life span substantially (Fig. 5C) and hastened the age-related loss of locomotor function (Fig. 5D). Expression of Sod2-IR viaMef2-Gal4 or 24B-Gal4 reduced SOD2 activity in whole-body extractswithout significantly impacting the activity of SOD1 (Fig. 6),confirming that expression of Sod2-IR knocks down the function ofSod2. Interestingly, the life span and locomotor effects of Sod2-IRexpression in the musculature via Mef2-Gal4 and 24B-Gal4 werecomparable to or possibly more severe than those of the ubiquitousdrivers Actin-Gal4 and da-Gal4 (Table 1).

We expressed Sod2-IR in cardiac muscle to determine whetherSod2 function in this tissue might be important for life span andlocomotor behavior across age. Median life spanwas reduced by ∼11%(Fig. 5E) and age-related loss of locomotor behavior was significantlyaccelerated (Fig. 5F) by expression of Sod2-IR driven by the cardiacGal4 line GMH5 [36]. The life span and locomotor effects of GMH5-

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driven Sod2-IR expression, however, were considerably less severethan those observed with Mef2-Gal4 and 24B-Gal4 (Table 1).

We examined lacZ reporter expression in whole-body adultcryosections to characterize the expression patterns for da-Gal4,Mef2-Gal4, and 24B-Gal4 (Figs. 7A, 7C, and 7E). Gal4 drivers oftenexhibit age-dependent patterns of expression [22,37] and could, inprinciple, be sensitive to the enhanced oxidative stress in flies withknockdown of Sod2. We therefore assessed da-Gal4, Mef2-Gal4, and24B-Gal4 expression patterns across the shortened adult life span offlies that concurrently expressed UAS-lacZ and UAS-Sod2-IR24transgenes. As expected [13,29], da-Gal4 drove expression of lacZthroughout the body (Fig. 7A). Mef2-Gal4 drove expression mainly inthe thoracic muscle (Fig. 7C), whereas 24B-Gal4 drove lacZ expression

Fig. 7. Spatial and temporal expression patterns of Gal4 in Sod2 knockdown flies. Gal4 expressa UAS-lacZ reporter. Whole-body sagittal cryosections stained for β-galactosidase activity forexpressed ubiquitously at all ages examined. (C)Mef2-Gal4 expressed strongly in indirect fligthe indirect flight muscle and fat body across age. Expression of 24B-Gal4 throughout the thsubstrate (E, d1 inset). lacZ expression driven by (B) da-Gal4, (D) Mef2-Gal4, and (F) 24B-Gpuparium formation (24 PPF), and adults at the indicated ages in days. There was a significapb0.0001, N=3 groups of three flies per genotype). Data in B, D, and F are means±SEM.

in thoracic muscle, abdominal muscle, and fat body (Fig. 7E and insetfor d1). This is largely consistent with the muscle-specific expressionpatterns previously reported for these two Gal4 drivers [34,38–40]. Atthe level of resolution possible with this approach, we did not observea substantive change in lacZ expression across the short life span ofSod2 knockdown flies driven by the three Gal4 drivers (Figs. 7A, 7C,and 7E). The spatial patterns of expression in da-Gal4,Mef2-Gal4, and24B-Gal4 drivers overlap in the musculature, but do not appreciablychange with age in short-lived Sod2 knockdown animals.

We quantified lacZ reporter activity inwhole-body extracts of Sod2knockdown animals throughout development and adulthood tocharacterize the temporal expression patterns of the da-Gal4, Mef2-Gal4, and 24B-Gal4 drivers. Whole-body lacZ reporter activity was

ionwas assessed in flies harboring the indicated Gal4 driver, a Sod2-IR24 transgene, and(A) da-Gal4, (C)Mef2-Gal4, and (E) 24B-Gal4 at the indicated ages in days. (A) da-Gal4ht muscle and to a lesser extent in other muscles across age. (E) 24B-Gal4 expressed inoracic muscle was observed upon incubating tissue sections for longer periods in X-galal4 was quantified in whole-body extracts of third-instar larvae (L3), pupae 24 h post-nt effect of age on Gal4 expression levels for all three Gal4 drivers (individual ANOVAs,

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dynamic across age for all three drivers. Expression of lacZ driven byda-Gal4 was easily detectable during late larval and pupal stages,increased to a peak of expression at day 7 of adulthood, and thendeclined by day 11 of adulthood (close to the maximum life span offlies expressing Sod2-IR ubiquitously) to a level similar to that foundin larvae and pupae (Fig. 7B). Mef2-Gal4 drove minimal lacZexpression during the larval and pupal stages, but thereafter drovevery strong expression in adults that increased out to the near-maximal life span of 7 days in flies expressing Sod2-IR via Mef2-Gal4(Fig. 7D). 24B-Gal4 expressed lacZ at moderate levels duringdevelopment with peaks of expression at days 1 and 7 of adulthood(Fig. 7F). Although the three Gal4 drivers have distinct temporalpatterns of expression, they share the common feature of robustexpression during adulthood.

To address whether normal expression of SOD2 in muscles islimiting for life span and age-related locomotor impairment, weassessed survival and negative geotaxis across age in flies over-expressing Sod2 using the Gal4 drivers Mef2 and 24B. Overexpression

Fig. 8. Knockdown of Sod2 causes mitochondrial pathology in indirect flight muscles (IFMs).day-old flies. (A and B) Control Sod2-IR24 alone. (C and D) Control 24B-Gal4 alone. (E, F, I, anindicated by mf and representative mitochondria are indicated by an asterisk in (A). IFMarrangement of cristae (I and J, ⁎⁎). Swollen mitochondria were often engulfed in autophag

of Sod2 in the musculature did not extend life span or ameliorate age-related locomotor impairment (data not shown). In fact, Sod2overexpression driven by Mef2-Gal4 resulted in a slight decrease inlife span and acceleration of age-related locomotor impairment.

Cellular consequences of Sod2 knockdown

To gain insight into the cellular consequences of knocking downSod2, we examined the ultrastructure of indirect flight muscle (IFM)by transmission electron microscopy (TEM). IFM ultrastructure wasindistinguishable at 1 (Figs. 8A and 8C) and 7 days (Figs. 8B and 8D) ofage in control flies. The IFMs in control animals at both ages had aregular arrangement of myofibrils interspersed by rows of denselypacked mitochondria. The IFMs from flies expressing Sod2-IR in themusculature via 24B-Gal4 (Fig. 8E) or Mef2-Gal4 (Fig. 8G) also hadlargely normal ultrastructure at 1 day of age, although there wereoccasional clusters of swollen mitochondria (Figs. 8I and 8J). Theswollen mitochondria in 1-day-old Sod2 knockdown flies exhibited a

Images are TEM analyses of IFMs in (A, C, E, G, I, and J) 1-day-old and (B, D, F, and H) 7-d J) 24B-Gal4;Sod2-IR24. (G and H)Mef2-Gal4;Sod2-IR24. Representative myofibrils ares of 1-day-old Sod2 knockdown flies contained swollen mitochondria with a rarifiedolysosomes (I, arrow).

Fig. 9. Sod2 knockdown in muscle causes loss of mitochondrial content, decreased ATP,and increased caspase activity. (A) Quantitation of mitochondrial content in IFMs at 1(d1) and 7 (d7) days of age in controls (Sod2-IR24, 24B-Gal4, or Mef2-Gal4 alone) orSod2 knockdown flies (Sod2-IR24;24B-Gal4 and Sod2-IR24;Mef2-Gal4). Two-wayANOVA indicated significant overall effects of age (pb0.001) and genotype (pb0.01)with an interaction (pb0.01) between these two factors (N=4–8 images pergenotype). Bonferroni's multiple comparison posttests revealed a significant reductionin mitochondrial content between controls and Sod2 knockdown flies at 7 days of age(⁎pb0.05). (B) Thoracic ATP content in control and Sod2 knockdown flies at 1 and 7days of age. Age (pb0.0001) and genotype (pb0.01) had significant effects on thoracicATP content (two-way ANOVA, N=3 groups of five flies per genotype). Sod2knockdown genotypes had significantly less ATP than controls at 7 days of age(Bonferroni's posttests, ⁎pb0.05). (C) Caspase activity (nM AMC/s/mg protein) incontrol (Sod2-IR24, Sod2-IR15, or Mef2-Gal4 alone) and Sod2 knockdown (Mef2-Gal4;Sod2-IR15 and Mef2-Gal4;Sod2-IR24) flies. Overall, caspase activity was significantlyelevated in both Sod2 knockdown genotypes relative to control flies (two-way ANOVA,pb0.0001, N=4 groups of two flies per genotype). Bonferroni's posttests revealed thatcaspase activity was increased in both Sod2 knockdown genotypes at all ages (pb0.05).Data (means±SEM) are representative of two independent experiments.

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rarified arrangement of cristae and in some cases were associatedwith lysosomes (Fig. 8I, arrow), indicating that the damagedmitochondria were being removed by autophagy. By 7 days of age,the IFMs in flies with Sod2 knockdown in muscle had an obviousreduction inmitochondrial content yet they retained normalmyofibrilultrastructure (Figs. 8F and 8H). Quantitative assessment of TEMimages revealed that, whereas mitochondrial content was maintainedin control flies out to 7 days of age, it was reduced by 32–49% in Sod2knockdown animals during this same time (Fig. 9A).

Ubiquitous disruption of Sod2 function through mutations in mice[11] and flies [14] or RNAi-mediated knockdown of Sod2 in flies [13]causes oxidative damage to and decreased function of mitochondrialenzymes important in energy metabolism. We measured ATP contentin extracts from Sod2 knockdown flies to determine whetherdecreased SOD2 activity in the musculature impaired mitochondrialfunction. We focused on ATP content in the thorax because this tissueis predominantly composed of muscle in Drosophila. ATP contentincreased significantly from day 1 to day 7 of adulthood in control fliesharboring a Sod2-IR transgene alone, 24B-Gal4 alone, or Mef2-Gal4alone (Fig. 9B) as expected from previous reports [41]. ATP content at1 day of age was indistinguishable in control flies and in flies withknockdown of Sod2 in the muscle (Fig. 9B). By 7 days of age, however,Sod2 knockdown flies had a 25–30% reduction in thoracic ATP contentcompared to age-matched controls (Fig. 9B).

Decreased SOD2 activity causes an elevation in apoptosis in mouseliver, retina, and heart [9,42–44] and also in the fly brain [14]. Wetherefore determinedwhether knockdown of Sod2 in the musculaturealtered caspase activity (a marker of apoptosis) and, if so, whethercaspase activity changed with age in Sod2 knockdown flies. Asexpected from previous studies [23], control flies had low levels ofcaspase activity in thoraces out to 4 days of age (Fig. 9C). In Sod2knockdown flies, caspase activity was significantly elevated at all agesexamined.

Discussion

SOD2 is a critical antioxidant localized to mitochondria [5].Whole-body loss of Sod2 function through RNAi-mediated silencingor knockout of the endogenous Sod2 gene dramatically shortens lifespan and causes behavioral defects in flies [12–15] and mice [6–8].Here, we used tissue-specific expression of Sod2-IR transgenes inDrosophila to address whether these effects on life span andbehavior are due to loss of Sod2 function throughout the body orwithin specific tissues. We focused on the nervous system and themusculature owing to their high metabolic activity with attendantgeneration of ROS [4].

Expression of Sod2-IR transgenes throughout the nervous system,in specific subsets of neurons required for negative geotaxis behavior,or in glia modestly shortened life span and accelerated age-relatedloss of negative geotaxis. The effects of nervous system expression ofSod2-IR, however, were considerably less severe than observed in flieswith whole-body Sod2-IR expression (Table 1). Although otherinterpretations are possible, these data suggest that the nervoussystemmay not be a key site of Sod2 functionwithin the context of lifespan and locomotor function across age in flies.

Consistent with the possibility that tissues outside of the nervoussystem are important for Sod2 function, expression of Sod2-IR in themusculature via two Gal4 lines (Mef2 and 24B) dramaticallyshortened life span and accelerated the age-dependent loss oflocomotor behavior (Table 1). The effect of Sod2-IR expression viathese two Gal4 drivers was equal to or even greater than the effect ofubiquitous Sod2 knockdown (Table 1). Importantly, we confirmedthat Mef2-Gal4 and 24B-Gal4 drive expression in the thoracic flightmuscle and found that both were expressed throughout the short lifespan of Sod2 knockdown flies. Together, these data indicate that Sod2

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functions within the musculature to protect flies from prematuredeath and loss of locomotor behavior.

Previous studies point toward Sod2 being important for normalmuscle function and integrity. For example, Sod2 knockout miceexhibit reduced activity of ETC components and lipid accumulation inskeletal muscle [7,11]. Additionally, Sod2 knockout mice display adilated cardiomyopathy [6,7]. These studies, however, do not explicitlyaddress the importance of Sod2 function in the muscle for whole-organism vitality and vigor. Our studies extend these previous reportsby demonstrating that normal life span and locomotor functiondepend on expression of Sod2 within the musculature in Drosophila.

The effects of Sod2 knockdown in the musculature led us topropose that overexpression of this enzyme in this tissue mightextend life span or preserve locomotor behavior across age.Overexpression of Sod2 in muscle, however, had no effect orpossibly even negative effects on these two measures. One possibleinterpretation of these data is that Sod2 in Drosophila muscle is notlimiting for life span or locomotor senescence. An extrapolation ofour findings is that the life-span-extending effect of ubiquitousSod2 overexpression [45] might be due to increased SOD2 activityin a tissue other than or in addition to the musculature. Our data,however, are also consistent with other interpretations. Forexample, increased longevity was obtained via ubiquitous SOD2overexpression conditionally during adulthood only [45], whereasin the Gal4 system used here, SOD2 overexpression occurs duringdevelopment and adulthood. Hence, it is possible that overexpres-sion of SOD2 during development has negative physiologicalconsequences that counteract any positive effects of SOD2 over-expression on aging during adulthood. Additionally, it is possiblethat overexpression of SOD2 beyond a level providing maximalbenefit might have negative consequences on aging. Because ROScan act as important signaling molecules [46] in addition to drivingoxidative damage, any negative outcomes associated with SOD2overexpression could be due to inhibition of ROS signaling.Additional experiments are required to address these possibilities.

Sod2 knockout mice exhibit dilated cardiomyopathy, a severedegenerative state thought to be an important contributor to theperinatal mortality in these animals [6,7]. Interestingly, patientswith hereditary hemochromatosis that carry a Sod2 allele associatedwith reduced enzymatic activity were found to be at a higher riskfor developing cardiomyopathy [47]. Although expression of Sod2-IRin Drosophila cardiac muscle shortened life span and accelerated theloss of locomotor function across age, these effects were rathermodest compared to knocking down Sod2 ubiquitously or in theflight muscle (Table 1). Hence, in flies, Sod2 function within cardiacmuscle is important for normal life span and locomotor functionacross age, but other muscles such as the thoracic flight muscle arelikely to be more important sites of SOD2 activity.

Sod2 knockout mice display swollen mitochondria in nervous andmuscle tissue [6,43,48], although this phenotype is not universallyobserved [7]. Knockdown of Sod2 in the musculature of flies causedmitochondrial swelling in 1-day-old animals that was followed by adecrease in bothmitochondrial and ATP content by 7 days of age. Loss ofSod2 in muscle, therefore, causes a progressive decrease in mitochon-drial functional capacity that manifests during adulthood in Drosophila.

There is a strong link between oxidative stress and apoptosis[49]. For example, exposure of cells or intact animals to pro-oxidantchemicals (i.e., exogenous oxidative stress) induces apoptosis[23,50,51]. Oxidative stress derived from endogenous sources canalso activate apoptosis. Sod2 knockout mice have elevated apoptosisin liver, retina, and heart [9,42,44,52] and Sod2 null flies haveincreased apoptosis in the central brain [14]. Our studies indicatethat knockdown of Sod2 in the musculature is sufficient to induce alarge increase in apoptosis in Drosophila, which could be animportant mechanism related to the short life span and possiblyother phenotypes in Sod2 knockdown flies. Additionally, our studies

support the notion that induction of apoptosis by knockdown ofSod2 can occur in a tissue-autonomous fashion and thereforedoes not require loss of Sod2 throughout the body.

Acknowledgments

This work was supported by Grant AG024259 from the NationalInstitutes for Aging (M.G.) and Grant MOP79519 from the Institute ofAging of the Canadian Institutes of Health Research (L.S.).

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