Neuronal expression of a single-subunit yeast NADH-ubiquinone oxidoreductase (Ndi1) extends Drosophila lifespan

Abstract

The 'rate of living' theory predicts that longevity should be inversely correlated with the rate of mitochondrial respiration. However, recent studies in a number of model organisms, including mice, have reported that interventions that retard the aging process are, in fact, associated with an increase in mitochondrial activity. To better understand the relationship between energy metabolism and longevity, we supplemented the endogenous respiratory chain machinery of the fruit fly Drosophila melanogaster with the alternative single-subunit NADH-ubiquinone oxidoreductase (Ndi1) of the baker's yeast Saccharomyces cerevisiae. Here, we report that expression of Ndi1 in fly mitochondria leads to an increase in NADH-ubiquinone oxidoreductase activity, oxygen consumption, and ATP levels. In addition, exogenous Ndi1 expression results in increased CO2 production in living flies. Using an inducible gene-expression system, we expressed Ndi1 in different cells and tissues and examined the impact on longevity. In doing so, we discovered that targeted expression of Ndi1 in fly neurons significantly increases lifespan without compromising fertility or physical activity. These findings are consistent with the idea that enhanced respiratory chain activity in neuronal tissue can prolong fly lifespan.

Figure 1. NDI1 can function in Drosophila mitochondria

All assays were performed on female flies between 6-10 days of age. A Targeted expression of NDI1 in fly mitochondria. The purity of cytoplasmic (C) and mitochondrial (M) fractions was confirmed using antibodies against Drosophila Porin 1 (dPorin1) and α-tubulin as mitochondrial and cytoplasmic markers, respectively. The expression of NDI1 protein in each fraction was examined by immunoblotting with polyclonal rabbit antisera against NDI1. Genotypes: Control 1: +/da-GAL4, Control 2: UAS-Ndi1/+; Ndi1: UAS-Ndi1/da-GAL4. B Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) was performed on mitochondrial proteins isolated from Ndi1-expressing flies and controls. Each lane represents increasing amount (5, 10, 20μg) of protein. Expression of NDI1 does not affect the assembly of the endogenous respiratory chain enzymes. Genotypes: Control: +/da-GAL4, Ndi1: UAS-Ndi1/da-GAL4. C. Representative polarographic trace showing rates of oxygen consumption in mitochondria isolated from Ndi1-expressing flies and controls. Ndi1 confers a 30% increase in glutamate/malate-stimulated (complex I-specific) respiration and promotes rotenone-insensitive respiration, without affecting succinate-stimulated (complex II-specific) respiration. Genotypes: Control: +/da-GAL4, Ndi1: UAS-Ndi1/da-GAL4.

Figure 2. Expression of Ndi1 can increase respiratory chain activity in Drosophila

Genotypes: Control: +/da-GAL4, Ndi1: UAS-Ndi1/da-GAL4. All assays were performed on female flies between 6-10 days of age. A Ndi1 confers a significant increase in NADH-ubiquinone oxidoreductase (complex I) enzymatic activity compared to controls (p=0.0007). Rotenone insensitive activity was measured after addition of 1 μM rotenone. B Ndi1-expressing flies display a significant increase in mean ATP levels compared to controls (p=0.004). C. Ndi1-expressing flies display a significant increase in CO₂ production compared to controls (P < 0.001). D. Expression of Ndi1 in fly mitochondria does not affect ROS production (p>0.05). A, B, D n = 3, C n=8. Error bars indicate standard deviation. Student's t-test was used.

Figure 3. Effects of tissue-specific expression of Ndi1 on fly longevity

Two independent UAS-Ndi1 lines (UAS-Ndi1¹ & UAS-Ndi1²), were crossed to GeneSwitch driver lines (A-B the ubiquitous Tubulin (tub)-GS driver, C-D the fat-body driver S₁106, E-F the pan-neuronal driver Elav-GS) and lifespan curves are shown as induced (2 mg/ml RU486 during development and 10 mg/ml RU486 from the onset of adulthood (black circles) or uninduced (−RU486, open circles). A. Lifespan curves of UAS-Ndi1¹/tub-GS females. A moderate decrease in survival was observed in response to RU486 (P=0.0441). B. Lifespan curves of UAS-Ndi1²/tub-GS females. A moderate increase in survival was observed in response to RU486 (P=0.018). C. Lifespan curves of UAS-Ndi1¹/S₁106 females. A 22% decrease in survival was observed in response to RU486 (p<0.0001). D. Lifespan curves of UAS-Ndi1²/S₁106 females. A 9% decrease in survival was observed in response to RU486 (p<0.0001). E. Lifespan curves of UAS-Ndi1¹/Elav-GS females. A 12% increase in survival was observed in response to RU486 (p<0.0001). F. Lifespan curves of UAS-Ndi1²/Elav-GS females. A 21% increase in survival was observed in response to RU486 (p<0.0001). Complete survival data on different RU486 concentrations are given in Tables S1, S2, S3. The significance of the difference between survival curves was analyzed using log-rank statistical test.

Figure 4. Neuronal expression of Ndi1 increases complex I enzymatic activity and ATP levels in heads of long-lived flies

A-E. UAS-Ndi1/Elav-GeneSwitch flies were fed with 2 μg/ml RU486 during development and 10 μg/ml RU486 during the adulthood stage; uninduced flies were fed with diluent. All assays were performed on female flies between 6-10 days of age. A. Increased Ndi1 mRNA in heads of long-lived flies (p<0.0001). B. Increased NADH-ubiquinone oxidoreductase (complex I) enzymatic activity in heads of long-lived flies compared to uninduced controls (p = 0.0029). Rotenone insensitive activity was measured after addition of 1μM rotenone. C. Increased ATP levels in heads of long-lived flies (p=0.0002). D. The expression and activities of endogenous respiratory chain enzymes are unchanged in heads of long-lived flies as assayed by Blue native-polyacrylamide gel electrophoresis (BN-PAGE) followed by in-gel activity staining. Coomasie Brilliant Blue (CBB) was used for estimating the amounts of the separated protein complexes. E. Neuronal expression of Ndi1 does not affect CO₂ production in living flies (p>0.05). A-D n = 3, E n=8. Error bars indicate standard deviation. Student's t-test was used.

Figure 5. Reduced levels of reactive oxygen species (ROS) in brains of Ndi1-expressing flies

A-B. UAS-Ndi1/Elav-GeneSwitch flies were fed with 2 μg/ml RU486 during development and 10 μg/ml RU486 during the adulthood stage; uninduced flies were fed with diluent. Assays were performed on 45 day-old female flies. A. Neuronal expression of Ndi1 reduces ROS abundance in fly brains detected by dihydroethidium. B. Quantification of the relative ROS levels in brains of Ndi1-expressing flies normalized to uninduced control brains; Ndi1 expression in neurons reduces ROS levels by 51% (p=0.001). n = 5 brains per treatment. Error bars indicate standard deviation. Student's t-test was used.

Figure 6. Ndi1 expressing flies are resistant to the endogenous complex I inhibitor rotenone and the free radical generator paraquat

A-C, UAS-Ndi1/Elav-GeneSwitch flies were fed with 2 μg/ml RU486 during development and 10 μg/ml RU486 during the adulthood stage; uninduced flies were fed with diluent. Survival curves are shown as induced (+RU486, solid circles) or uninduced (-RU486, open circles). All assays were performed on female flies between 6-10 days of age. A Ndi1 confers tolerance to 250 μM rotenone (p< 0.0001) and B 30 mM paraquat (p< 0.0001), but does not affect C starvation resistance (p=0.9278). The significance of the difference between survival curves was analyzed using the log-rank statistical test.

Figure 7. Long-lived Ndi1-expressing flies display normal fertility and physical activity

UAS-Ndi1/Elav-GeneSwitch flies were fed with 2 μg/ml RU486 during development and 10 μg/ml RU486 during the adulthood stage; uninduced flies were fed with diluent. A. Average number of progeny produced from crosses between 5 wild-type males and 5 UAS-Ndi1/Elav-GeneSwitch-GAL4 females fed RU486 or diluent. Long-lived Ndi1 expressing flies display normal fertility compared to uninduced controls (p>0.05; two-way Analysis of Variance). B. Activity of 6- 10 day-old flies recorded with Drosophila Activity Monitor over a 24 hour period is shown. Long-lived Ndi1 expressing flies display normal physical activity compared to uninduced controls (p>0.05; two-way Analysis of Variance).