Increased longevity mediated by yeast NDI1 expression in Drosophila intestinal stem and progenitor cells - PubMed (original) (raw)

Increased longevity mediated by yeast NDI1 expression in Drosophila intestinal stem and progenitor cells

Jae H Hur et al. Aging (Albany NY). 2013 Sep.

Abstract

A functional decline in tissue stem cells and mitochondrial dysfunction have each been linked to aging and multiple aging-associated pathologies. However, the interplay between energy homeostasis, stem cells, and organismal aging remains poorly understood. Here, we report that expression of the single-subunit yeast alternative NADH dehydrogenase, ndi1, in Drosophila intestinal stem and progenitor cells delays the onset of multiple markers of intestinal aging and extends lifespan. In addition, expression of ndi1 in the intestine increases feeding behavior and results in organismal weight gain. Consistent with increased nutrient uptake, flies expressing ndi1 in the digestive tract display a systemic reduction in the activity of AMP-activated protein kinase (AMPK), a key cellular energy sensor. Together, these results demonstrate that ndi1 expression in the intestinal epithelium is an effective strategy to delay tissue and organismal aging.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1. Intestine-specific expression of ndi1 increases lifespan

(A) Analysis of NDI1 enzymatic activity in mitochondria isolated from intestines. NDI1 is expressed by transgenic expression of an ndi1 cDNA under control of the intestine-specific TIGS-2 driver (TIGS-2>ndi1). Transgenic expression is induced by exposure of flies to the drug RU486 (100mg/l). Expression of ndi1 is sufficient to confer flavone sensitive, rotenone insensitive NADH dehydrogenase activity to mitochondria isolated from intestines. (***p<0.001, t test, 5 replicates per condition, mitochondria from 10 dissected intestines from female flies per replicate). (**B**) Survival curves of female _TIGS-2_>ndi1 flies with or without RU486-mediated transgene induction. Constitutive expression of ndi1 by RU486 exposure (10mg/l during development, 50mg/l during adulthood) increases lifespan (p<0.0001, log-rank test, at least 200 flies per condition). (**C**) Survival curves of female _esgGAL4_>ndi1 flies compared to isogenic controls. UAS-ndi1 and the isogenic control strain (w1118) were crossed to esgGAL4. A 50% increase in mean survival was observed in response to ndi1 expression (p<0.0001, log-rank test, at least 200 flies per condition). (**D**) Survival curves of female _5961GS_>ndi1 flies with or without RU486-mediated transgene induction. Adult-onset expression of ndi1 by RU486 exposure (0.5mg/l) increases fly lifespan (p<0.0001, log-rank test, at least 200 flies per condition).

Figure 2. ndi1 expression maintains intestinal homeostasis during aging

(A) Immunofluorescence images evaluating intestinal homeostasis during aging. Control flies (esgGAL4_>+, upper panel) and ndi1 expressing flies (esgGAL4_>ndi1, lower panel) were assayed for esg+ cells (GFP+ cells) and mitotic cells (pHH3+ cells, arrows) 10 days and 50 days post eclosion. Scale bars=50μm. (B) Quantification of proportion of esg+ cells. The proportion of esg+ cells (GFP+ cells) in all cells (DAPI stain) was increased in aged control flies (“+”, esgGAL4>+), but not in ndi1 expressing flies (“_ndi1_”, esgGAL4>ndi1). (*p<0.05, ***p<0.001, One-way ANOVA with Tukey's post hoc test, at least 22 flies per condition). (**C**) Quantification of mitotic cells per field of view. The median number of mitotic events (pHH3+ cells per field of view) is elevated in aged control flies (“_+_”, _esgGAL4_>+), but not in ndi1 expressing flies (“_ndi1_”, esgGAL4>ndi1). (*p<0.05, **p<0.01, ***p<0.001, Kruskal-Wallis test followed by Dunn's multiple comparisons, at least 22 flies per condition). (**D**)Quantification of ROS levels per area in ISCs/EBs. _ndi1_ expression (“_ndi1_”, _esgGAL4>ndi1) decreases DHE fluorescence within esg+ cells(GFP+ cells, arrows in figure S2A) in both 10 and 30 day old intestines compared to isogenic controls (“+”, esgGAL4>+).(*p<0.05, **p<0.01, t test, at least 3 images per gut, 10 guts per condition). (**E**) Quantification of ROS levels per area in midguts. _ndi1_ expression in ISCs/EBs (“_ndi1_”, _esgGAL4>ndi1) results in decreased DHE fluorescence in gut tissues relative to isogenic controls (“_+”, esgGAL4>+) at 30 days post eclosion. (***p<0.001, t test, at least 3 images per gut, 10 guts per condition). (**F**) Proportion of flies showing loss of intestinal integrity as a function of age, assayed using blue dye no. 1. Aged flies that express _ndi1_ in ISCs/EBs (_esgGAL4>ndi1) show reduced levels of intestinal barrier dysfunctionrelative to controls (esgGAL4>+). (***p<0.001, binomial test, at least 190 flies per condition). (**G**) Proportion of flies showing loss of intestinal integrity as a function of age in 5961GS_>ndi1 flies with or without RU486-mediated transgene induction. Adult-onset expression of ndi1 by RU486 exposure (0.5mg/l) improves maintenance of intestinal integrity during aging. (H) Systemic expression of Drosomycin, Drosocin and Diptericin in 10 and 45 day old flies. Aged flies that express ndi1 in ISCs/EBs (“_ndi1_”, esgGAL4>ndi1) show reduced expression of antimicrobial peptides (AMPs) relative to controls (“+_”, esgGAL4>+). (**p<0.01, ***p<0.001, t test, 5 replicates per condition, 5 flies per replicate).

Figure 3. ndi1 expression in the intestine stimulates feeding behavior

(A) Analysis of total food consumption using a capillary feeding (CAFE) assay. Flies expressing ndi1 in ISCs/EBs (esgGAL4>ndi1) consume significantly more food relative to controls (esgGAL4>+). (*p<0.05, **p<0.01, t test, 10 replicates per condition, 10 flies per replicate). (**B**) Analysis of feeding proportion using a colorimetric dye-tracking assay. Flies that express ndi1_ in ISCs/EBs (_esgGAL4>ndi1) had a significantly greater proportion of flies that fed during the assay period relative to controls (esgGAL4>+). (***p<0.001, binomial test, approximately 90 flies per condition). (**C**) Analysis of meal size using a colorimetric dye-tracking assay (constitutive ndi1_ expression). Of those flies that ate during the assay period in (B), meal size was significantly greater in flies that express ndi1 in ISCs/EBs (_esgGAL4>ndi1) relative to controls (esgGAL4>+). (*p<0.05, ***p<0.001, t test, 50-95 flies that ate from B per condition). (**D**) Analysis of total food consumption using a CAFE assay in 5961GS>ndi1 flies with or without RU486-mediated transgene induction. Ten days of induced ndi1 expression in ISCs/EBs of adults by exposure to RU486 (0.5mg/l) increases total food consumption relative to uninduced controls. (*p<0.05, t test, 6 replicates per condition, 10 flies per replicate). (**E**) Analysis of meal size using a colorimetric dye-tracking assay in 5961GS>ndi1 flies with or without RU486-mediated transgene induction. Ten days of induced ndi1 expression in ISCs/EBs of adults by exposure to RU486 (0.5mg/l) increases meal size relative to uninduced controls. (*p<0.05, t test, approximately 85 flies that ate during the assay period in Figure S3A). (**F**) Analysis of intestinal function by assaying excreta shape. The proportion of oblong deposits (RODs, Figure S3E, arrow) in excreta is significantly lower in deposits from flies expressing ndi1_ in ISCs/EBs (_esgGAL4>ndi1) relative to controls (esgGAL4>+). (*p<0.05, ***p<0.001, binomial test, at least 180 deposits per condition). (**G**) Analysis of intestinal function by excreta pH. Flies expressing ndi1_ in ISCs/EBs (_“ndi1”, esgGAL4>ndi1) had more alkaline excreta relative to controls (“+”, esgGAL4>+) at day 10 of adulthood in colorimetric analyses of excreta pH after feeding on BPB medium. (**p<0.01, ***p<0.001, t test, at least 180 deposits per condition). (**H**) Weights of flies with constitutive ndi1_ expression in ISCs/EBs as a function of age. Flies that express ndi1 in ISCs/EBs (_“ndi1”, esgGAL4>ndi1) are heavierthan isogenic controls (“+”, esgGAL4>+) and maintain their weight through day 30 of adulthood. (**p<0.01, **p<0.01, ***p<0.001, t test, 12 replicates per condition, 5 flies per replicate). (**I**) Weights of 5961GS>ndi1 flies with or without RU486-mediated transgene induction. Ten days of adulthood only induction of ndi1 in ISCs/EBs by exposure to RU486 (0.5mg/l) is sufficient to significantly increase weight relative to uninduced controls. (**p<0.01, t test, 6 replicates per condition, 10 flies per replicate). (**J**) Protein content as a function of age. Protein content is significantly decreased in control flies (“+”, esgGAL4>+) at 30 days of adulthood, but is maintained in flies expressing ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1). (*p<0.05, **p<0.01, t test, 4 replicates per condition, 5 flies per replicate). (**K**) Triacylglyceride content as a function of age. Thin-layer chromatography (Figure S3G) and densitometry for triacylglyceride content show a significant decrease in control flies (“+”, esgGAL4>+) at 30 days of age whereas flies that express ndi1 in ISCs/EBs (“ndi1”, esgGAL4>ndi1) maintain triacylglyceride stores with age. (**p<0.01, t test, 5 replicates per condition, 5 flies per replicate).

Figure 4. ndi1 expression in the intestine produces alterations in systemic metabolic signaling pathways

(A-B) Western blot (A, Figure S4A) and densitometric analysis (B) of AMPKα phosphorylation at Thr184. AMPKα phosphorylation (normalized to a loading control, beta-Actin)is significantly decreased in flies that express ndi1 in ISCs/EBs (“ndi1_”, esgGAL4>ndi1), relative to isogenic controls (“+”, esgAL4>+) at day 10 of adulthood. (*p<0.05, t test, 5 replicates per condition, 15 flies per replicate). (**C**) Transcript levels of downstream targets of dFOXO. _4E-BP, InR, l(2)efl_, and _ImpL2_ transcript levels are significantly lower in flies that express _ndi1_ in ISCs/EBs (“_ndi1_”, _esgGAL4>ndi1) relative to isogenic controls (“_+”, esgAL4>+) at day 10 of adulthood. (**p<0.01, t test, 5 replicates per condition, 5 flies per replicate). (**D-E**) Western blot (D, Figure S4B) and densitometric analysis (**E**) of AKT phosphorylation at Ser505 or Thr423, and total AKT levels. AKT phosphorylation (normalized to total AKT) is not altered in flies that express _ndi1_ in ISCs/EBs (“_ndi1_”, _esgGAL4>ndi1) relative to isogenic controls (“_+”, esgAL4>+) at day 10 of adulthood. Total AKT levels (normalized to beta-Actin) are also unchanged. (n.s., t test, 5 replicates per condition, 5 flies per replicate). (F) Transcript levels of head dilp genes. dilp2, dilp3, and dilp5 transcript levels from heads of flies that express ndi1 in ISCs/EBs (“_ndi1_”, esgGAL4>ndi1) are significantly lower than those of controls (“+”, esgAL4>+) at day 10 of adulthood. (*p<0.05, **p<0.01, ***p<0.001, t test, 5 replicates per condition, 30 heads per replicate). (**G-H**) Immunofluorescence staining (**G**) and quantification (**H**) of DILP2 levels in insulin producing cells (IPCs). DILP2 fluorescence in IPCs of flies that express _ndi1_ in ISCs/EBs (“_ndi1_”, _esgGAL4>ndi1) are significantly lower compared to that of controls (“_+”, esgAL4>+) at day 10 of adulthood. (*p<0.05, t test, at least 170 IPCs from 12 different brains). (**I-J**) Transcript levels of _sNPF_ (**I**) and its cognate receptor, sNPFR1 (**J**) in heads. Flies that express _ndi1_ in ISCs/EBs (“_ndi1_”, _esgGAL4>ndi1) have significantly lower sNPF and sNPFR1 transcript levels in heads than controls (“_+_”, esgAL4>+) at day 10 of adulthood. (*p<0.05, **p<0.01, t test, 4-5 replicates per condition, 30 heads per replicate).

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Increased longevity mediated by yeast NDI1 expression in Drosophila intestinal stem and progenitor cells - PubMed (original) (raw)