Abstract
The theory that accumulation of reactive oxygen species (ROS) in internal organs is a major promoter of aging has been considered negatively. However, it is still controversial whether overexpression of superoxide dismutases (SODs), which remove ROS, extends the lifespan in Drosophila adults. We examined whether ROS accumulation by depletion of Cu/Zn-SOD (SOD1) or Mn-SOD (SOD2) influenced age-related impairment of the nervous system and muscles in Drosophila. We confirmed the efficient depletion of Sod1 and Sod2 through RNAi and ROS accumulation by monitoring of ROS-inducible gene expression. Both RNAi flies displayed accelerated impairment of locomotor activity with age and shortened lifespan. Similarly, adults with nervous system-specific depletion of Sod1 or Sod2 also showed reduced lifespan. We then found an accelerated loss of dopaminergic neurons in the flies with suppressed SOD expression. A half-dose reduction of three pro-apoptotic genes resulted in a significant suppression of the neuronal loss, suggesting that apoptosis was involved in the neuronal loss caused by SOD silencing. In addition, depletion of Sod1 or Sod2 in musculature is also associated with enhancement of age-related locomotion impairment. In indirect flight muscles from SOD-depleted adults, abnormal protein aggregates containing poly-ubiquitin accumulated at an early adult stage and continued to increase as the flies aged. Most of these protein aggregates were observed between myofibril layers. Moreover, immuno-electron microscopy indicated that the aggregates were predominantly localized in damaged mitochondria. These findings suggest that muscular and neuronal ROS accumulation may have a significant effect on age-dependent impairment of the Drosophila adults.
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Acknowledgments
We acknowledge D. Bohmann (Rochester university) and Bloomington Stock Center for providing the fly stocks. This study was partially supported by Grant-in-Aid for Scientific Research A (22241052).
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Saori Oka and Jun Hirai have equally contributed.
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Supplementary Fig. 1. Lifespan curves of adult males with depleted SOD1 or SOD2 by induction of each dsRNA using a pan-neuronal or a pan-muscular Gal4 driver. (A) Lifespan curves of SOD-depleted adult males by induction of each dsRNA using elav-Gal4 driver. SOD-depleted flies (elav > Sod1RNAi IR4, elav > Sod1RNAi F103, elav > Sod2RNAi IR15), controls (WT(Canton S) and elav-Gal4/Y). The curves were plotted using Kaplan–Meier survival analysis. The lifespan of SOD1-depleted adults (elav > Sod1RNAi IR4) is significantly shorter than that of controls (more than a 100 flies were tested, p < 0.00625). The lifespan shortening observed in elav > Sod1RNAi F103 is not statistically significant (p < 0.03). (B) Lifespan curves of SOD-depleted adult males by induction of each dsRNA using Mef2-Gal4 driver. SOD-depleted flies (Mef2 > Sod1RNAi IR4, Mef2 > Sod1RNAi F103, Mef2 > Sod2RNAi IR15, Mef2 > Sod2RNAi HMS00783), controls (WT(Canton S) and Mef2-Gal4/+). The curves were plotted using Kaplan–Meier survival analysis. The lifespan of SOD1-depleted or SOD2-depleted adults (Mef2 > Sod1RNAi IR4, Mef2 > Sod2RNAi IR15, Mef2 > Sod2RNAi HMS00783) is significantly shorter than that of controls (more than a 100 flies were tested, p < 0.00625). We performed the log-rank test for each pair of control and RNAi type under the significance level (0.05/8 = 0.00625) by Bonferroni’s correction. Supplementary Fig. 2. Nervous system-specific depletion of Sod1 or Sod2 resulted in the loss of DA neurons in adult brains. (A, B) Induced expression of Sod1 (A) or Sod2 (B) dsRNA using a pan-neuronal driver, elav-Gal4 resulted in significantly lower number of DA neurons in adult brains, compared with that in the control male flies from controls (WT(Canton S) or elav-Gal4 driver stock) (10 flies were tested for each genotype). (A) SOD1-depleted flies (elav > Sod1RNAi IR4, elav > Sod1RNAi F103), controls (WT(Canton S) and elav-Gal4/+). We used ANOVA followed by the Student’s t-test and the significance level was set at 0.05/2 = 0.025 under Bonferroni’s correction (*means p < 0.025). In adult brains from elav-Gal4 > UAS-Sod1RNAi F103 males, the significant decrease of DA neurons was observed in PAL, PPL1 clusters. In adults with less efficient depletion of SOD1 (elav-Gal4 > UAS-Sod1RNAi IR4), the significant reduction appeared in the PPL1 cluster. (B) SOD2-depleted flies (Mef2 > Sod2RNAi IR15), controls (WT(Canton S) and Mef2-Gal4/+). In addition, the significant decrease in DA neurons was observed in PAL cluster in elav-Gal4 > UAS-Sod2RNAi IR15 male brains. We used the Student’s t-test and the significance level was set at 0.05 (**means p < 0.05). Supplementary Fig. 3. Depletion of Sod1 and Sod2 by induction of each dsRNA exclusively at adult stage also resulted in the loss of DA neurons. (A, B) Induced expression of Sod1 (A) or Sod2 (B) dsRNA by the DJ703 Gal4 driver that becomes active in adult stage also resulted in a lower number of DA neurons in the brains as compared with that in the control male flies (WT(Canton S) and DJ703/+) (10 flies were examined for each genotype). (A) SOD1-depleted flies (DJ703 > Sod1RNAi IR4, DJ703 > Sod1RNAi F103), controls (WT(Canton S) and DJ703/+). We used ANOVA followed by the Student’s t-test and the significance level was set at 0.05/2 = 0.025 under Bonferroni’s correction (*means p < 0.025). The reduction of DA neurons is significant in the PPL1 cluster from DJ703 > UAS-Sod1RNAi IR4 and DJ703 > UAS-Sod1RNAi F103 flies. (B) SOD2-depleted flies (DJ703 > Sod2RNAi IR15), controls (WT(Canton S) and DJ703/+). The significant reduction appeared in the PAL cluster in SOD2-depleted adult brains (DJ703 > UAS-Sod2RNAi IR15) (10 flies were examined for each genotype). We used the Student’s t-test and the significance level was set at 0.05 (*means p < 0.05). Supplementary material 1 (PDF 239 kb)
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Oka, S., Hirai, J., Yasukawa, T. et al. A correlation of reactive oxygen species accumulation by depletion of superoxide dismutases with age-dependent impairment in the nervous system and muscles of Drosophila adults. Biogerontology 16, 485–501 (2015). https://doi.org/10.1007/s10522-015-9570-3
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DOI: https://doi.org/10.1007/s10522-015-9570-3