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

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.

(e) Survival curves of daGS>UAS-Drp1 males with or without RU486-mediated transgene induction from day 30 onwards. The shaded area indicates the duration of Drp1 induction.
(k) Survival curves of 5966GS>UAS-Drp1 females with or without RU486-mediated transgene induction from day 30 onwards. The shaded area indicates the duration of Drp1 induction.
(l) Survival curves of daGS>UAS-Drp1 females with or without RU486 feeding from day 30 to day 37. The shaded area indicates the duration of RU486 feeding. p<0.0001, log rank test; n > 182 flies.
(m) Survival curves of daGS>w 1118 females with or without RU486 feeding from day 1 to day 30. The shaded area indicates the duration of RU486 feeding. P>0.05, log rank test; n > 236 flies.
(n) Survival curves of daGS>w 1118 females with or without RU486 feeding from day 30 onwards. The shaded area indicates the duration of RU486 feeding. P>0.05, log rank test; n > 241 flies.
(o) Survival curves of daGS>w 1118 females with or without RU486 feeding from day 30 to day 37. The shaded area indicates the duration of RU486 feeding. P>0.05, log rank test; n > 239 flies.
(p) QPCR analyses of Drp1 mRNA levels on day 37 in daGS>w 1118 females with or without RU486 feeding from day 30 to day 37. n = 5 replicates with 3 flies per replicate; p > 0.05 and is non-significant (n.s.); two-tailed unpaired t-test. feeding. n = 8 vials of 10 flies per condition; p > 0.05 and is non-significant (n.s.); two-tailed unpaired t-test.
(b and c) Spontaneous activity graph (b) of 37 day old daGS>w 1118 females with or without RU486 feeding as measured by DAM system beam breaks. Quantification of total activity per 8 fly per hour (c) from spontaneous activity graphs. n = 3 vials of 10 flies per condition; p > 0.05 and is non-significant (n.s.); two-tailed Mann Whitney U test. (d) Starvation survival curves of daGS>w 1118 females with or without RU486 feeding from day 30 onwards. p > 0.05; log rank test; n = 100 flies.
(e) Fecundity time course of daGS>w 1118 females with or without RU486 feeding since day 30 onwards. n = 360 flies on day 10; p > 0.05 and is non-significant (n.s.); one-way ANOVA/Bonferroni's multiple comparisons test.
(d and e) Immunostaining of indirect flight muscles (d) from day 37 daGS>w 1118 females with or without RU486 feeding for 7 days from day 30 to day 37 showing mitochondria (green channel, anti ATP5a) and muscles (red channel, rhodamine staining for F-actin). Scale bar is 5µm. Quantification of mitochondrial size (e); n = 8; p > 0.05 is non-significant (n.s.); twotailed unpaired t-test.
(f and g) Indirect flight muscles (f) from day 37 daGS>w 1118 females with or without feeding from day 30 onwards showing TMRE staining as a marker for mitochondrial membrane potential. Quantification of mitochondrial membrane potential (g) measured by TMRE staining as shown in (f). n = 8-12 flies; p > 0.05 is non-significant (n.s.) ; two-tailed unpaired t-test.
Boxplots (b 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. Bars (c and g) depict mean ± S.D. (a) In situ respirometry of permeabilized muscle bundles from 10 and 37 day old daGS>UAS-Drp1 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 replicates with 2 thoraces per replicate; ***p < 0.001 and **p < 0.01; two-tailed unpaired ttest. (b) In situ respirometry of permeabilized muscle bundles from 37 day old daGS>UAS-Drp1 females with or without RU486 feeding from day 3 onwards 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 replicates with 2 thoraces per replicate; p > 0.05 is non-significant (n.s.); two-tailed unpaired t-test.

Supplementary Figure 4. RU486 does not impact mitochondrial complex I activity or ROS levels in control flies
(c) In situ respirometry of permeabilized heads from 37 day old daGS>UAS-Drp1 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 = 5-6 replicates with 10 heads per replicate; *p < 0.05; two-tailed unpaired t-test.
(d) Quantification of free superoxide radicals from staining of indirect flight muscles from 10 and 37 day old daGS>UAS-Drp1 females. Staining was done for mitochondria (Mitotracker green staining) and levels of superoxide radicals (staining with MitoSOX™ reagent oxidation of which produce red fluorescence when it interacts with superoxide radicals). n = 9 replicates; p > 0.05 is non-significant (n.s.); two-tailed unpaired t-test.
(e) Quantification of marker of mitochondrial activity in 10 and 37 day old daGS>w 1118 females with or without RU486 feeding for 7 days from day 30 to day 37. Complex I activity measurement in isolated mitochondrial pellet from 10 and 37 day old adult females. n = 5 replicates with 8 flies per replicate; *p < 0.05 and p > 0.05 is non-significant (n.s.); one-way ANOVA/Bonferroni's multiple comparisons test.
(b and c) Immunostaining of indirect flight muscles (b) from day 37 daGS> w 1118 females with or without RU486 feeding from day 30 to day 37, 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). Scale bar is 5µm. Quantification of mitochondrial ds-DNA (c) in muscles (as shown in b); n = 6 flies; p > 0.05 and is non-significant (n.s.); two-tailed unpaired t-test. (d) Western blot detection of mitochondrial fusion-promoting factor Mitofusin in isolated mitochondria from day 37 daGS>UAS-Drp1 females with or without RU486 feeding from day 30 to day 37.
(e-f) Immunostaining of indirect flight muscles (e) from day 37 daGS> w 1118 females with or without RU486 feeding from day 30 to day 37 showing mitochondria (green channel, anti ATP5a) and an autophagic marker (red channel, anti-ATG8a). Scale bar is 5µm. Quantification (f) of ATG8a foci co-localizing with mitochondria (as shown in e); n = 6 flies; p > 0.05 and is non-significant (n.s.); two-tailed unpaired t-test.