Promoting Drp1-mediated mitochondrial fission in midlife prolongs healthy lifespan of Drosophila melanogaster

Abstract

The accumulation of dysfunctional mitochondria has been implicated in aging, but a deeper understanding of mitochondrial dynamics and mitophagy during aging is missing. Here, we show that upregulating Drp1-a Dynamin-related protein that promotes mitochondrial fission-in midlife, prolongs Drosophila lifespan and healthspan. We find that short-term induction of Drp1, in midlife, is sufficient to improve organismal health and prolong lifespan, and observe a midlife shift toward a more elongated mitochondrial morphology, which is linked to the accumulation of dysfunctional mitochondria in aged flight muscle. Promoting Drp1-mediated mitochondrial fission, in midlife, facilitates mitophagy and improves both mitochondrial respiratory function and proteostasis in aged flies. Finally, we show that autophagy is required for the anti-aging effects of midlife Drp1-mediated mitochondrial fission. Our findings indicate that interventions that promote mitochondrial fission could delay the onset of pathology and mortality in mammals when applied in midlife.Mitochondrial fission and fusion are important mechanisms to maintain mitochondrial function. Here, the authors report that middle-aged flies have more elongated, or 'hyper-fused' mitochondria, and show that induction of mitochondrial fission in midlife, but not in early life, extends the health and life of flies.

Fig. 1

Midlife Drp1 induction extends lifespan. a, b Immunostaining of indirect flight muscles a from 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction for 7 days from day 30 to day 37, showing mitochondria (green channel, anti-ATP5a) and Drp1 (red channel, anti-Drp1). Scale bar is 5 µm. Quantification of mitochondrial size b; n = 7 individual thoraces; ***p < 0.001; two-tailed unpaired t-test. c Immunostaining of optic lobe/brain from 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction for 7 days from day 30 to day 37, showing mitochondria (green channel, anti-ATP5a) and nuclei (blue channel, TO-PRO-3). Scale bar is 5 µm. d Survival curves of daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 1 to day 30. The shaded area indicates the duration of Drp1 induction. p = 0.42, log-rank test; n > 179 flies. e Survival curves of daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 onwards. The shaded area indicates the duration of Drp1 induction. p < 0.0001, log-rank test; n > 179 flies. f Survival curves of daGS > UAS-dMfn-RNAi females with or without RU486-mediated transgene induction from day 30 onwards. The shaded area indicates the duration of dMfn RNAi. p < 0.0001, log-rank test; n > 175 flies. g Survival curves of daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. The shaded area indicates the duration of Drp1 induction. p < 0.0001, log-rank test; n > 291 flies. h qPCR analyses of Drp1 mRNA levels on day 44 in daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 5 biological replicates with three individual flies per replicate; ***p < 0.001 and **p < 0.01; one-way ANOVA/Bonferroni’s multiple comparisons test. Boxplots b display the first and third quartile, with the horizontal bar at the median and whiskers showing the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Bars h depict mean ± s.d. RU486 was provided in the media at a concentration of 25 μg/ml for a–e, g, h and 50 μg/ml for f

Fig. 2

Midlife Drp1 induction delays age-onset pathology and prolongs healthspan. a Capillary feeding assay (CAFE) of 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 8 vials of 10 flies per condition; p > 0.05 and is non-significant (n.s.); two-tailed unpaired t-test. b, c Spontaneous physical activity b of 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. Quantification of total activity per fly per hour c from spontaneous activity graphs. n = 3 vials of 10 flies per condition; **p < 0.01; two-tailed unpaired t-test. d Climbing index as a measure of endurance of 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 90 flies per condition; ^∗ p < 0.05; two-tailed Mann–Whitney test. e Survival curves without food of 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. p < 0.01; log-rank test; n = 100 flies. f Whole body lipid stores of 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 5 biological replicates with three flies per replicate; *p < 0.05; two-tailed unpaired t-test. g Fecundity of daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 630 flies on day 10; *p < 0.05; one-way ANOVA/Bonferroni’s multiple comparisons test. h Intestinal integrity during aging of daGS > UAS-Drp1 females with or without RU486-mediated transgene induction since midlife (day 30) onwards. n = 448 flies on day 10; **p < 0.01, *p < 0.05; one-way ANOVA/Bonferroni’s multiple comparisons test. Bars a, c and d depict mean ± s.d. and bars g, h depict mean ± s.e.m. Boxplots f display the first and third quartile, with the horizontal bar at the median and whiskers showing the most extreme data point, which is no more than 1.5 times the interquartile range from the box. RU486 was provided in the media at a concentration of 25 μg/ml

Fig. 3

Midlife Drp1 induction rejuvenates mitochondrial morphology and function. a–d Immunostaining of indirect flight muscles from day 10, 28 and 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37 showing mitochondria (green channel, anti-ATP5a) and muscles (red channel, rhodamine staining for F-actin). Scale bar is 5 µm. e–h Electron micrograph of indirect flight muscles from day 10, 28 and 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37, showing mitochondria and muscles. Scale bar is 1 µm. i–l Staining of indirect flight muscles from day 10, 28, and 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37 showing TMRE fluorescence. Scale bar is 5 µm. m Quantification of mitochondrial size in indirect flight muscles from day 10, 28, and 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37 as shown in a–d. n = 4–7 flies; ***p < 0.001, **p < 0.01; one-way ANOVA/Bonferroni’s multiple comparisons test. n Quantification of relative mitochondrial size in indirect flight muscles from day 10, 28, and 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37 as shown in e–h. Data are represented as mitochondrial fractions in percentages per size. n = 3 flies per condition. o Quantification of mitochondrial membrane potential measured by TMRE staining as shown in i–l from day 10, 28, and 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 10–19 flies; ***p < 0.001, **p < 0.01; Kruskal–Wallis test/Dunn’s multiple comparisons test. Boxplots m display the first and third quartile, with the horizontal bar at the median and whiskers showing the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Bars o depict mean ± s.e.m. RU486 was provided in the media at a concentration of 25 μg/ml

Fig. 4

Midlife Drp1 induction improves mitochondrial respiratory function. a, b Quantification of markers of mitochondrial activity in 28, 37, and 44 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction since midlife (day 30 onwards). Complex I activity measurements a in isolated mitochondrial pellet from 28 to 37 day old adult females. n = 5 biological replicates with five flies per replicate; **p < 0.01; two-tailed unpaired t-test. b In situ respirometry of permeabilized muscle bundles from 37 to 44 day old adult females to assess the capacity for oxidative phosphorylation (OXPHOS) and Electron Transport System (ETS) flux, and the flux control ratio of Complex I by rotenone inhibition. n = 6–8 biological replicates with 2 flies per replicate; **p < 0.01 and *p < 0.05; one-way ANOVA/Bonferroni’s multiple comparisons test. c, d Staining of indirect flight muscles c from 37 day old daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37, showing mitochondria (green channel, Mitotracker green staining) and levels of superoxide radicals (red channel, staining with MitoSOX reagent). Scale bar is 5 µm. Quantification of free superoxide radicals d; n = 11–16 biological replicates; **p < 0.01; two-tailed unpaired t-test. e qPCR analyses of Hsp60, Hsp10, and mtHsp70 (Hsc70-5) on day 37 in daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. n = 5 biological replicates with 3 flies per replicate; p > 0.05 and is non-significant (n.s.); two-tailed unpaired t-test. Bars a depict mean ± s.d. Boxplots b, d and e display the first and third quartile, with the horizontal bar at the median and whiskers showing the most extreme data point, which is no more than 1.5 times the interquartile range from the box. RU486 was provided in the media at a concentration of 25 μg/ml

Fig. 5

Midlife Drp1 induction improves proteostasis in aged flies. a–d Immunostaining of indirect flight muscles from day 10, 28, and 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37, showing protein polyubiquitinated aggregates (red channel, muscles stained with phalloidin/F-actin and green channel, antipolyubiquitin). e–h Immunostaining of indirect flight muscles from day 10, 28, and 37 daGS > UAS-Drp1 ^K38A females with or without RU486-mediated transgene induction from day 30 to day 37, showing protein polyubiquitinated aggregates (red channel, muscles stained with phalloidin/F-actin and green channel, antipolyubiquitin). i Quantification of polyubiquitin aggregates in muscle as shown in a–d. n = 9–11 flies; ***p < 0.001; one-way ANOVA/Bonferroni’s multiple comparisons test. j Quantification of polyubiquitin aggregates in muscle as shown in e–h. n = 9–14 flies; ***p < 0.001; one-way ANOVA/Bonferroni’s multiple comparisons test. k–m Immunostaining of optic lobe/brain from day 10 and day 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37, showing protein polyubiquitinated aggregates (red channel stained with phalloidin/F-actin; green channel, antipolyubiquitin and blue channel, nuclei stained with TO-PRO-3). k’–m’ Insets from k–m, respectively. n Quantification of polyubiquitin aggregates in optic lobe/brain as shown in k, m. n = 6–13 flies; ***p < 0.001; one-way ANOVA/Bonferroni’s multiple comparisons test. o, p Western blot o detection of total ubiquitin-conjugated proteins in isolated mitochondria from day 37 daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to day 37. Densitometry of ubiquitin blots p from mitochondrial pellet. n = 6 replicates (25 flies per replicate); ***p < 0.001; two-tailed Mann–Whitney test. Boxplots i, j and n display the first and third quartile, with the horizontal bar at the median and whiskers showing the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Bars p depict mean ± s.d. Scale bar is 5 µm. RU486 was provided in the media at a concentration of 25 μg/ml

Fig. 6

Midlife Drp1 induction facilitates mitophagy in aged flies. All experiments were carried out in daGS > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 onwards. a, b Western blot a detection of mitochondrial respiratory complex subunits in thoraces dissected from day 37 flies. Densitometry of blots b; n = 4 replicates with 5 thoraces per replicate; *p < 0.05; two-tailed unpaired t-test. c, d Immunostaining of indirect flight muscles c from day 37 flies showing mitochondrial DNA (green channel, anti-ds DNA antibody), nuclear DNA (blue channel, stained with TO-PRO-3) and muscles (red channel, stained with phalloidin/F-actin). Quantification of mitochondrial ds DNA d in muscles (as shown c); n = 4 flies; *p < 0.05 two-tailed unpaired t-test. e, f Western blot e detection of mitochondrial fusion-promoting factor Mitofusin in isolated mitochondria from day 37 flies. Densitometry of blots f; n = 3 replicates with 20 flies per replicate; **p < 0.01 two-tailed unpaired t-test. g, h Immunostaining of indirect flight muscles g from day 33 flies showing mitochondria (green channel, anti-ATP5a) and Atg8a (red channel, anti-Atg8a). Quantification h of Atg8a foci co-localizing with mitochondria (as shown in g); n = 7 flies; ***p < 0.001; two-tailed unpaired t-test. i–l Immunostaining of indirect flight muscles from day 37 flies showing mitochondria (green channel, anti-ATP5a) and p62 (red channel, anti-p62). Quantification l of P62 foci per muscle area (as shown in i–k. n = 7–9 flies; **p < 0.01 and ***p < 0.001; one-way ANOVA/Bonferroni’s multiple comparisons test. m, n Western blot m detection of P62 levels in isolated mitochondria from day 37 flies. Densitometry of blots n; n = 3 replicates, 20 flies per replicate; *p < 0.05; two-tailed unpaired t-test. Bars b, d, f and n depict mean ± s.d. Boxplots h and l display the first and third quartile, with the horizontal bar at the median and whiskers showing the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Scale bar is 5 µm. RU486 was provided in the media at a concentration of 25 μg/ml

Fig. 7

Autophagy is required for Drp1-mediated longevity. All experiments, except e, were carried out on day 37 in daGS, UAS-Atg1-RNAi > UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 to 37. a qPCR analyses of Atg1 and Drp1 mRNA levels. n = 5 replicates (three flies per replicate); *p < 0.05, **p < 0.01; two-tailed Mann–Whitney test. b–d Immunostaining b of indirect flight muscles showing mitochondria (green channel, anti-ATP5a) and Atg8a foci (red channel, anti-Atg8a). Quantification of mitochondrial size c and total number of Atg8a foci d; n = 6–10 flies; **p < 0.01, ***p < 0.001; two-tailed unpaired t-test. e Survival curves with or without RU486-mediated transgene induction from day 30 onwards. The shaded area indicates the duration of Drp1 activation and Atg1 RNAi. p = 0.96, log-rank test; n > 288 flies. f In situ respirometry of permeabilized muscles to assess the capacity for oxidative phosphorylation (OXPHOS) and Electron Transport System (ETS) flux, and the flux control ratio of Complex I by rotenone inhibition. n = 6 replicates (two thoraces per replicate); n.s. indicates not significant; two-tailed unpaired t-test. g, h Staining of indirect flight muscles g showing mitochondria (green channel, Mitotracker green staining) and levels of superoxide radicals (red channel, staining with MitoSOX reagent). Quantification of free superoxide radicals h; n = 12 replicates; n.s. indicates not significant; two-tailed unpaired t-test. i, j Immunostaining of indirect flight muscles j showing protein polyubiquitinated aggregates (red channel, muscles stained with phalloidin/F-actin and green channel, antipolyubiquitin). i Quantification of polyubiquitin aggregates in muscle (as shown j); n = 16 flies; **p < 0.01; two-tailed unpaired t-test. k, l Western blot k detection of total ubiquitin-conjugated proteins in isolated mitochondria. Densitometry of ubiquitin blots l from mitochondrial pellet; n = 8 replicates, 25 flies per replicate; *p < 0.05; two-tailed unpaired t-test. Bars a, d and l depict mean ± s.d. Boxplots c, f, h and i display the first and third quartile, with the horizontal bar at the median. Scale bar is 5 µm. RU486 was provided in the media at a concentration of 25 μg/ml