PMC

Knock-down of Sod2 causes mitochondrial pathology in indirect flight muscles (IFMs). Images are TEM analysis of IFM in 1-day-old (A, C, E, G, I and J) and 7-day-old (B, D, F and H) flies. (A and B), control Sod2-IR24 alone. (C and D), control 24B-Gal4 alone. (E, F, I and J), 24B-Gal4; Sod2-IR24. (G and H), Mef2-Gal4; Sod2-IR24. Representative myofibrils are indicated by mf and representative mitochondria are indicated by an asterisk (*) in panel A. IFMs of 1-day-old Sod2 knock-down flies contained swollen mitochondria with a rarified arrangement of cristae (I and J, **). Swollen mitochondria were often engulfed in autophagolysosomes (I, arrow).

Gal4 drivers for neurons involved in negative geotaxis. There was a significant overall effect of genotype (i.e. Gal4 driver used) on behavior (one-way ANOVA, p < 0.0001, n = 4–5 (representing 100–125 flies) for Gal4;Shi^ts1 flies, n = 35 (representing 875 flies) for Shi^ts1/+ control). (A) Expression of Shi^ts1 via the Gal4 drivers 91Y, 188Y, Appl, D42 and OK307 caused a temperature-sensitive disruption of negative geotaxis (Tukey HSD multiple comparison, p < 0.05). (B) Negative geotaxis was not significantly affected by temperature in flies expressing Shi^ts1 via all other Gal4 drivers (Tukey HSD multiple comparison). Data (mean ± S.E.M., compiled from 7 experiments) are the percent negative geotaxis observed at 25°C remaining at 31°C.

Expression of Sod2-IR in muscle reduces life span and accelerates age-related locomotor impairment. Two independent Sod2-IR transgenes, Sod2-IR15 and Sod2-IR24, were expressed via Mef2-Gal4 (A and B), 24B-Gal4 (C and D), or GMH5-Gal4 (E and F). (A, C and E) Life span was significantly shortened by expression of Sod2-IR via all three Gal4 drivers (log-rank test, p < 0.0001, n = 150 flies per genotype). (B, D and F) Age-related locomotor impairment was accelerated in flies with Sod2-IR expression driven by all three Gal4 lines (two-way ANOVA; effect of age and genotype, p < 0.0001; Tukey’s HSD multiple comparison, p < 0.05; n = 5 vials of 25 flies per genotype). Data are representative of two independent experiments.

Ubiquitous expression of Sod2 RNAi reduces SOD2 activity, shortens life span and accelerates locomotor senescence. (A) Protein extracts (45 μg total protein/lane) from adult males (~2 days old) were electrophoresed on native polyacrylamide gels. SOD activity was visualized using an “in-gel” assay as described in 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-IR24 transgenes alone (log-rank test, p < 0.0001, n = 150 flies per genotype). (C) Age-related loss 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, p < 0.0001, n = 5 vials of 25 adult males per genotype). Negative geotaxis data are mean ± SEM.

Nervous system Sod2-IR expression has modest effects on locomotor senescence and life span. Sod2-IR expression via the pan-neuronal drivers 188Y-Gal4 (A), 91Y-Gal4 (B), Appl-Gal4 (C) or elav-Gal4 (D) significantly impaired negative geotaxis behavior across age (individual two-way ANOVAs for effect of age and genotype, p < 0.0001 for both factors, n = 5–10 vials of 25 males per genotype). Tukey’s honestly significant difference (HSD) post-test revealed that all Sod2 knock-down lines performed significantly worse across age than either of their control groups (p < 0.05). Negative geotaxis data (mean ± SEM) are compiled from two independent experiments except in (D) which is from one experiment. Life span was also reduced in flies (150 per genotype) following Sod2-IR expression via 188Y-Gal4 (E) or elav-Gal4 (F). See for percent decrease in median life span.

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 gel assay described in materials and methods. Each lane contained 45 μg total protein from adult males (~2 days old). Flies had the 24B-Gal4, Mef2-Gal4 or no Gal4 driver along 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 activity was significantly reduced in flies with Sod2-IR24 and Sod2-IR15 expression driven by 24B-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 post-test, * p < 0.05). (C) Quantitation of whole-body SOD1 activity. Whole-body SOD1 activity was not affected by genotype (individual one-way ANOVAs, not significant, n = 3 groups of 25 flies per genotype). Data in B and C are mean ± SEM.

Neuronal Sod2 knock-down impairs locomotor function across age and shortens survival. Sod2-IR24 was expressed specifically in motor neurons via D42-Gal4 (A and B), giant fiber neurons via OK307-Gal4 (C and D) or c17-Gal4 (E and F), dopaminergic neurons via TH-Gal4 (G) and throughout glia via repo-Gal4 (H and I). Negative geotaxis performance across age (A, C, E, G, I) was significantly worse in flies expressing Sod2-IR24 via all Gal4 drivers except OK307-Gal4 in (C) (individual two-way ANOVAs for effect of age and genotype, p<0.0001, n = 5–10 vials of 25 male flies for both factors in all data sets, followed by Tukey’s HSD post-test (p < 0.05, or n.s. for OK307-Gal4;Sod2-IR24). Life span (B,D,F and I) was significantly reduced in all Sod2 knock-down genotypes (log-rank test p ≤ 0.0263, 150 flies per genotype). Survival was not assessed for Sod2-IR24 expression via TH-Gal4.

Sod2 knock-down in muscle causes loss of mitochondrial content, decreased ATP and increased caspase activity. (A) Quantitation of mitochondrial content in IFMs at 1 day (black bars) and 7 days (open bars) of age in controls (Sod2-IR24, 24B-Gal4 or Mef2-Gal4 alone) or Sod2 knock-down flies (Sod2-IR24; 24B-Gal4 and Sod2-IR24; Mef2-Gal4). Two-way ANOVA indicated significant overall effects of age (p < 0.001) and genotype (p < 0.01) with an interaction (p < 0.01) between these two factors (n = 4–8 images per genotype). Bonferroni’s multiple comparison post-tests revealed a significant reduction in mitochondrial content between controls and Sod2 knock-down flies at 7 days of age (* p < 0.05). (B) Thoracic ATP content in control and Sod2 knock-down flies at 1 (black bars) and 7 (open bars) days of age. Age (p < 0.0001) and genotype (p < 0.01) had significant effects on thoracic ATP content (two-way ANOVA, n = 3 groups of 5 flies per genotype). Sod2 knock-down genotypes had significantly less ATP than controls at 7 days of age (Bonferroni’s post-tests, * p < 0.05). (C) Caspase activity (nM AMC/sec/mg protein) in control (Sod2-IR24, Sod2-IR15 or Mef2-Gal4 alone) and Sod2 knock-down flies (Mef2-Gal4; Sod2-IR15 and Mef2-Gal4; Sod2-IR24) flies. Overall, caspase activity was significantly elevated in both Sod2 knock-down genotypes relative to control flies (two-way ANOVA, p < 0.0001, n = 4 groups of 2 flies per genotype). Bonferroni’s post-tests revealed that caspase activity was increased in both Sod2 knock-down genotypes at all ages (p< 0.05). Data (mean ± SEM) are representative of 2 independent experiments.

Spatial and temporal expression patterns of Gal4 in Sod2 knock-down flies. Gal4 expression was assessed in flies harboring the indicated Gal4 driver, a Sod2-IR24 transgene and a UAS-lacZ reporter. Whole-body sagittal cryosections stained for β-galactosidase activity (blue) for da-Gal4 (A), Mef2-Gal4(C) and 24B-Gal4 (E) at the indicated ages in days. (A) da-Gal4 expressed ubiquitously at all ages examined. (C) Mef2-Gal4 expressed strongly in indirect flight muscle and to a lesser extent in other muscles across age. (E) 24B-Gal4 expressed in the indirect flight muscle and fat body across age. Expression of 24B-Gal4 throughout thoracic muscle was observed upon incubating tissue sections for longer periods in X-gal substrate (E, D1 panel inset). lacZ expression driven by da-Gal4 (B), Mef2-Gal4(D) and 24B-Gal4 (F) were quantified in whole-body extracts of 3^rd instar larvae (L3), pupae 24 hrs post-puparium formation (24 PPF) and adults at the indicated ages in days. There was a significant effect of age on Gal4 expression levels for all three Gal4 drivers (individual ANOVAs, p < 0.0001, n = 3 groups of 3 flies per genotype). Data in B, D and F are mean ± SEM.