Rapamycin enhances survival in a Drosophila model of mitochondrial disease

Abstract

Pediatric mitochondrial disorders are a devastating category of diseases caused by deficiencies in mitochondrial function. Leigh Syndrome (LS) is the most common of these diseases with symptoms typically appearing within the first year of birth and progressing rapidly until death, usually by 6-7 years of age. Our lab has recently shown that genetic inhibition of the mechanistic target of rapamycin (TOR) rescues the short lifespan of yeast mutants with defective mitochondrial function, and that pharmacological inhibition of TOR by administration of rapamycin significantly rescues the shortened lifespan, neurological symptoms, and neurodegeneration in a mouse model of LS. However, the mechanism by which TOR inhibition exerts these effects, and the extent to which these effects can extend to other models of mitochondrial deficiency, are unknown. Here, we probe the effects of TOR inhibition in a Drosophila model of complex I deficiency. Treatment with rapamycin robustly suppresses the lifespan defect in this model of LS, without affecting behavioral phenotypes. Interestingly, this increased lifespan in response to TOR inhibition occurs in an autophagy-independent manner. Further, we identify a fat storage defect in the ND2 mutant flies that is rescued by rapamycin, supporting a model that rapamycin exerts its effects on mitochondrial disease in these animals by altering metabolism.

Keywords: Drosophila; complex I; leigh syndrome; mitochondria; rapamycin.

Figure 1. TOR inhibition by rapamycin increases lifespan in ND2 mutant flies

A. ND2 mutant flies exhibit increased phosphorylation of the TORC1 target p70S6k as compared to WT. Rapamycin abolishes TORC1 signaling. Actin is shown as control and quantification of biological triplicates is shown below. Significance was determined by ANOVA *p < .05 **p < 0.01. B. ND2 mutant flies exhibit a significant reduction in lifespan (red dashed line) in comparison to WT flies (black dashed line). Rapamycin treatment robustly extends lifespan in mtND2 flies (red solid line) as well as WT flies (black solid line). C. Quantification of median lifespan. ND2 flies have a 38% reduction in median lifespan compared to WT flies, and rapamycin robustly increases median lifespan in mtND2 flies by 34.2%. WT flies treated with rapamycin show a more modest increase in lifespan of 14.5%. D. ND2 mutant flies exhibit a mechanical-stress induced paralysis (solid black bars). Time to recovery after mechanical stress worsens with age and is not affected by rapamycin treatment (hashed black bars). WT flies show no paralysis, and only ND2 flies are shown. E. 15-day-old ND2 mutant flies exhibit heat-induced paralysis that is unaffected by rapamycin treatment. WT flies did not paralyze in the timeframe of this experiment; only ND2 flies are shown.

Figure 2. Effects of rapamycin on response to oxygen stress

A. ND2 mutant flies take longer to recover from anoxia-induced paralysis compared to WT controls. Rapamycin partially rescues this phenotype, but not to WT levels. Data are represented as mean +/- SEM. Significance was determined by ANOVA ***p < 0.001. B. Survival in 99.9% O₂. ND2 mutant flies are more susceptible to hyperoxia (solid red line) and rapamycin has no effect on response to hyperoxic stress (dashed red line).

Figure 3. Rapamycin extends lifespan in ND2 mutant flies in an autophagy-independent manner

A. QPCR on Atg1 mRNA levels. ND2 mutant flies that are heterozygous for the Atg1RNAi transgene but not the Gal4 driver show decreased levels of Atg1 mRNA as compared to WT. Expression of Atg1 RNAi driven by Hsp70-Gal4 driver results in a further decrease in Atg1 mRNA levels. Data are represented as mean value normalized to geometric mean of three housekeeping genes +/- SEM. B. Western blot analysis of the autophagic marker Atg8. Upper band is Atg8a (non-lipidated form) and lower band is Atg8b (lipidated form). Accumulation of the lipidated form (Atg8b) as seen in flies expressing Atg1RNAi and the Hsp70Gal4 driver (Lanes 3,4,7 and 8) is associated with a blockage in autophagy [39]. *p < 0.05, † 0.05 < p < 0.1. C. Lifespan analysis of WT and ND2 flies that are heterozygous for the Atg1 RNAi transgene alone (WT: black, ND2: red), or heterozygous for the Atg1 RNAi transgene and the Hsp70-Gal4 driver (WT: Green, ND2: Blue) on vehicle supplemented control food (dashed lines) or food supplemented with 200uM rapamycin (solid lines). Knockdown of autophagy in WT flies (yellow dashed line) abolishes the lifespan extension by rapamycin in WT flies (yellow solid line, p > .0.05). ND2 flies expressing Atg1 RNAi (dashed blue line) exhibit a significant decrease in lifespan compared to ND2 flies (dashed red line, ***:p < .001), but still prove responsive to rapamycin (solid blue line, ***:p < 0.001). D. Quantification of median lifespan.

Figure 4. ND2 mutant flies exhibit defects in fat metabolism that are reversed by rapamycin

A. Average weight per fly. 25-day-old ND2 flies exhibit a decrease in body mass that is rescued to WT levels by treatment with rapamycin. B. Free triglyceride levels. ND2 flies show decreased levels of free triglycerides that are rescued by treatment with rapamycin. C. Total triglyceride levels per mg of fly weight. ND2 mutant flies also exhibit a decrease in total triglyceride levels that are significantly increased by rapamycin treatment. Data are represented as mean +/- SEM. Significance was determined by ANOVA *p < .05, **p < 0.01, ***p < 0.001.