Midlife Drp1 induction prolongs lifespan and healthspan

In previous work, we showed that long-lived Parkin overexpressing flies display an increase in mitochondrial fission18. To test for a causal role for mitochondrial fission in lifespan extension, we sought to independently promote mitochondrial fission by upregulating Drp1 in aging flies. To do so, we used the ubiquitous daughterless-Gene-Switch (daGS) driver line to activate a UAS-Drp1 transgene created by30. Western blot and immunofluorescence analysis confirmed a dose-dependent and RU486- dependent induction of the Drp1 transgene (Supplementary Fig. 1a–d). Next, we confirmed that Drp1 induction confers a shift in mitochondrial dynamics toward increased fission in adult flies in both muscle and brain tissue (Fig. 1a–c). Next, we set out to examine the impact of temporally-defined shifts in mitochondrial dynamics on fly lifespan. Upregulation of Drp1 in early adulthood (days 0–30) or throughout adulthood had no significant impact on longevity (Fig. 1d, Supplementary Table 1). However, upregulating Drp1 from midlife (day 30) onwards significantly extended lifespan in both male and female flies (Fig. 1e, Supplementary Fig. 1e, f; Supplementary Table 1). Using an independently generated UAS-Drp1 transgene31, we confirmed that midlife Drp1 induction prolongs lifespan (Supplementary Fig. 1g; Supplementary Table 1). To further examine the impact of manipulating mitochondrial dynamics in midlife, we examined the impact of RNAi of Drosophila (d)Mfn on lifespan. Importantly, we observed that RNAi of dMfn from midlife onwards also extends both mean and maximum lifespan (Fig. 1f; Supplementary Table 1). Taken together, our findings demonstrate that promoting mitochondrial fission/inhibiting fusion from midlife onwards prolongs fly lifespan. To further validate our findings, we examined the impact of inhibiting mitochondrial fission/promoting mitochondrial fusion in midlife on fly lifespan. Importantly, we observed that expression of a dominant-negative Drp1 (Drp1K38A) transgene30 from midlife onwards shortens lifespan (Supplementary Fig. 1h; Supplementary Table 1) and upregulation of dMfn in midlife also shortens lifespan (Supplementary Fig. 1i; Supplementary Table 1). To refine the tissue-specific requirements involved in Drp1-mediated lifespan extension, we used the pan-neuronal Elav–Gene-Switch (ElavGS) driver line to increase Drp1 expression specifically in neurons32 and 5966-GS33 to increase Drp1 gene expression in the intestine. Upregulating Drp1 specifically in neurons or the intestine from midlife onwards is sufficient to prolong fly lifespan (Supplementary Fig. 1j, k; Supplementary Table 1).

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 Full size image

To refine the temporal requirements involved, we tested whether a transient induction of Drp1 was sufficient to prolong lifespan. We found that short-term induction of Drp1, from day 30 to 37, produced a robust increase in both mean and maximum lifespan (Fig. 1g, Supplementary Fig. 1l; Supplementary Table 1). Importantly, we find that Drp1 mRNA levels do not remain elevated following short-term midlife induction (Fig. 1h). RU486 had no impact on mitochondrial morphology, Drp1 mRNA levels or longevity in control flies when fed at any time period (Supplementary Figs 1m–p and 3d, e). Because ubiquitous, midlife Drp1 induction in female flies resulted in the most pronounced extension of lifespan, this paradigm was used in all further experiments.

To better understand the impact of Drp1-mediated mitochondrial fission in midlife on organismal health, we examined a number of markers of healthspan and behavior in aged flies following 7 days of Drp1 induction. As a reduction in food intake can modulate lifespan, we tested whether midlife Drp1 induction affects feeding behavior. Using a capillary feeding assay34, we failed to observe alterations in feeding behavior upon short-term, midlife Drp1 induction (Fig. 2a). It is important to determine whether interventions that prolong lifespan also extend healthspan or simply prolong a period of frailty. Short-term, midlife Drp1 induction conferred an increase in spontaneous physical activity (Fig. 2b and c). Importantly, this increase in physical activity was confined to day-time activity, as opposed to night time restlessness. Moreover, short-term, midlife Drp1 induction produced a significant improvement in an endurance exercise paradigm (Fig. 2d). The ability to withstand extrinsic stress is a marker of organismal health that declines with age. Short-term, midlife Drp1 induction improved survival when maintained on an agar-only diet to induce starvation in aged flies (Fig. 2e). This improvement in starvation resistance was linked to increased triglyceride (TAG) stores (Fig. 2f). Several interventions that promote longevity, such as dietary restriction, are frequently associated with reproductive tradeoffs35. In contrast, short-term, midlife Drp1 induction increased fecundity in aged flies (Fig. 2g).

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 Full size image

Aging is associated with a loss of tissue homeostasis, resulting in a decline in organ function. Recent work has shown that loss of intestinal barrier function is an evolutionarily conserved biomarker of aging36,37,38,39,40. To determine whether promoting mitochondrial fission in midlife can delay the onset of intestinal pathology, we examined intestinal barrier function in aging flies with and without midlife Drp1 induction. Loss of intestinal integrity can be assayed in living flies by monitoring the presence of non-absorbed dyes outside of the digestive tract post-feeding36, 41 (Fig. 2h). Remarkably, we observed a delay in the onset of intestinal barrier dysfunction upon Drp1 induction from day 30 onwards (Fig. 2h), indicating a delay in intestinal aging at the tissue level. Collectively, these data demonstrate that promoting Drp1-mediated mitochondrial fission in midlife improves healthspan and delays the onset of pathology linked to aging. Consistent with these findings, we observed that inhibiting mitochondrial fission or promoting mitochondrial fusion in midlife accelerates age-onset pathology. More specifically, either Drp1K38A expression (Supplementary Fig. 2g) or upregulation of dMfn (Supplementary Fig. 2h) in midlife confers early-onset intestinal barrier dysfunction. Feeding RU486 to control flies did not alter feeding behavior, spontaneous physical activity, loss of intestinal integrity, fecundity, or starvation sensitivity (Supplementary Fig. 2a–f).

Midlife Drp1 induction improves mitochondrial function in aged flies

To better understand the interplay between mitochondrial dynamics and aging, we used immunofluorescence (IF) microscopy and electron microscopy (EM) to examine age-related alterations in mitochondrial morphology in flight muscle. Using both approaches, we observed that in midlife (day 28) (Fig. 3b, f) the mitochondria are more elongated and less circular than in young muscle tissue (10 day old) (Fig. 3a, e). This shift in mitochondrial morphology becomes more pronounced at day 37 (Fig. 3c, g) suggesting an increase in fusion/decrease in fission. To examine the generality of this finding, we examined age-related alterations in mitochondrial morphology in an independent control laboratory strain, w dahomey, and confirmed a midlife shift toward a more elongated mitochondrial morphology in aged flight muscle (Supplementary Fig. 3a, b). To determine whether this age-related shift in mitochondrial dynamics was linked to altered expression of Drp1, we examined Drp1 expression levels during aging. We observed that an age-related decline in Drp1 expression accompanies the shift toward a more elongated, less fragmented mitochondrial morphology (Supplementary Fig. 3c).

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 Full size image

To gain insight into whether this shift in mitochondrial morphology is linked to altered mitochondrial function, we examined mitochondrial membrane potential using the potentiometric dye TMRE. Importantly, we observe that the accumulation of elongated mitochondria is linked to a significant decrease in TMRE fluorescence in aged flight muscle (Fig. 3i–k). Remarkably, we find that short-term, midlife induction (day 30–37) of Drp1 restores mitochondrial morphology, including cristae ultrastructure, to a more youthful state (compare Fig. 3d with 3a–c and Fig. 3h with 3e–g); quantification in Fig. 3m, n). Furthermore, we find that midlife induction of Drp1 restores a fragmented mitochondrial network, containing highly active mitochondria reminiscent of young tissue (compare Fig. 3l with 3i–k; quantification Fig. 3o). Together, these findings indicate that the midlife shift in mitochondrial morphology, toward more elongated, less circular mitochondria, contributes to the accumulation of depolarized mitochondria in aged flight muscle. Critically, upregulating Drp1 expression in midlife, for 7 days, rejuvenates mitochondrial morphology and function. RU486 had no impact on mitochondrial morphology or TMRE fluorescence in control flies (Supplementary Fig. 3d–g).

Next, we set out to examine the impact of midlife Drp1 induction on additional markers of mitochondrial health and function in aged flight muscle. First, we observed a significant increase in complex I enzymatic activity following short-term (7 days) induction of Drp1 (Fig. 4a). To build upon this finding, oxidative phosphorylation (OXPHOS) and electron transport system (ETS) capacities were measured, in permeabilized muscle bundles in situ, using high-resolution respirometry. We observed a decline in OXPHOS activity and ETS capacity in aged flies (Supplementary Fig. 4a). Upregulation of Drp1 throughout adulthood did not significantly improve mitochondrial respiratory activity (Supplementary Fig. 4b). However, promoting Drp1-mediated mitochondrial fission from midlife onwards resulted in an increase in OXPHOS capacity at day 44, and an increase in overall ETS capacity and the flux control ratio for Complex I on days 37 and 44 (Fig. 4b). In addition, we validated that promoting Drp1-mediated mitochondrial fission from midlife onwards improved mitochondrial function in brain tissue. More specifically, we observed that 7 days of Drp1 induction in midlife (day 30–37) resulted in an increase in OXPHOS capacity, an increase in overall ETS capacity and the flux control ratio for Complex I in head samples (Supplementary Fig. 4c). Next, we set out to determine whether short-term, midlife Drp1 induction impacts mitochondrial reactive oxygen species (ROS) levels. Following 7 days of Drp1 induction in midlife, we observed reduced mitochondrial ROS levels in aged flight muscle (Fig. 4c, d, Supplementary Fig. 4d). Taken together, our data show that promoting mitochondrial fission in midlife improves multiple markers of mitochondrial function and reduces mitochondrial ROS levels. RU486 had no significant effect on Complex I enzymatic activity or mitochondrial ROS levels in control flies (Supplementary Fig. 4e–g).

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 Full size image

In recent years, considerable attention has been paid to the concept of mitohormesis42. Indeed, studies in diverse organisms have shown that certain perturbations that impair mitochondrial function can promote longevity through mechanisms that may involve elevated mitochondrial ROS and induction of the mitochondrial unfolded protein response (UPRmt)43,44,45,46,47. As noted above, however, short-term, midlife Drp1 induction leads to reduced mitochondrial ROS levels (Fig. 4c, d). In addition, we failed to detect an induction of UPRmt genes following midlife Drp1 induction (Fig. 4e). These data indicate that midlife Drp1-mediated improvements in mitochondrial function and organismal health are not mediated via mitohormesis. RU486 had no impact on UPRmt gene expression in control flies (Supplementary Fig. 4h).

Midlife Drp1 induction improves proteostasis in aged flies

Mitochondrial dysfunction and loss of protein homeostasis (proteostasis) are two key hallmarks of aging1. However, the relationship between these two hallmarks of aging is not well understood. As noted above, we find that promoting Drp1-mediated mitochondrial fission in midlife improves mitochondrial function in aged flies. To determine whether midlife Drp1 induction could impact proteostasis in aged animals, we characterized the deposition of protein aggregates in aged flight muscle by IF microscopy. As previously reported18, 48, we observed that Drosophila flight muscles accumulate aggregates of ubiquitinated proteins during aging (Fig. 5a–c), consistent with a loss of proteostasis. Remarkably, short-term, midlife induction (days 30–37) of Drp1 resulted in reduced levels of protein aggregates in aged muscle (Fig. 5d, quantification in Fig. 5i) and aged brain tissue (Fig. 5k–m, quantification in Fig. 5n). To further validate our findings, we examined the impact of inhibiting mitochondrial fission/promoting mitochondrial fusion in midlife on proteostasis. Importantly, we observed that expressing a dominant-negative Drp1 (Drp1K38A) transgene from midlife onwards impairs proteostasis (Fig. 5e–h, j) and upregulation of dMfn in midlife also impairs proteostasis (Supplementary Fig. 5a, b).

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 Full size image

Recently, it has been reported that there is an over-representation of mitochondrial proteins in the insoluble protein fraction from aged animals49, 50. Interestingly, we observed that short-term, midlife Drp1 induction leads to reduced levels of ubiquitinated proteins in the mitochondrial fraction from aged flies (Fig. 5o, p, Supplementary Fig. 5e–g). Taken together, our findings reveal that the midlife shift toward more elongated mitochondria, in flight muscle, contributes to age-onset proteotoxicity. Critically, short-term, midlife induction of Drp1 improves proteostasis in aged animals. RU486 had no significant impact on the accumulation of protein aggregates in control flies (Supplementary Fig. 5c, d).

Midlife Drp1 induction facilitates mitophagy in aged flies

To further understand the impact of promoting mitochondrial fission in midlife on mitochondrial homeostasis, we examined markers of mitochondrial content following Drp1 induction. Short-term, midlife induction of Drp1 (days 30–37) resulted in lower levels of some respiratory chain subunit proteins (Fig. 6a, b and Supplementary Fig. 6a) and lower mitochondrial DNA levels in aged flight muscle (Fig. 6c, d). Furthermore, short-term midlife induction of Drp1 correlated with lower levels of dMfn, a degradation target of Parkin8, in the mitochondrial fraction from aged flies (Fig. 6e, f and Supplementary Fig. 6d). A possible explanation for these findings is that midlife Drp1 induction facilitates mitophagy and/or promotes selective turnover of some respiratory chain subunits. Indeed, recent work has shown that some mitochondrial respiratory chain proteins appear to be selectively routed for autophagosomal degradation by the PINK1-Parkin pathway51. To test the idea that midlife Drp1 induction facilitates mitophagy, we examined aged flight muscle for co-localization of autophagy markers with mitochondria. Short-term, midlife Drp1 induction (day 30–33) resulted in a significant increase in the co-localization of Atg8a/LC3 with mitochondria (Fig. 6g, h). In mammalian cells, the ubiquitin-binding autophagy adaptor protein p62/SQSTM1 accumulates on dysfunctional mitochondria52 and has been proposed to facilitate recruitment of damaged mitochondria to autophagosomes6. Furthermore, recent work has shown that Refractory to Sigma P, ref(2)P, the single Drosophila orthologue of p62 plays a critical role in the PINK1-Parkin mitophagy pathway53, 54. Impaired autophagy is associated with increased levels of p62 in mammals and Drosophila 55, indicating that levels of p62 reflect autophagic status. To gain further insight into age-related changes in mitochondrial homeostasis, we examined ref(2)P co-localization with mitochondria in aging flight muscle. We observed a striking accumulation of ref(2)P co-localized with mitochondria in midlife (from day 28 to day 37), consistent with a decline in mitophagy in aged muscle (Fig. 6i, j). Importantly, short-term, midlife induction of Drp1 (day 30–37) prevented the accumulation of ref(2)P co-localized with mitochondria during aging (compare Fig. 6j, k and quantification of p62 levels in Fig. 6l). To confirm this finding, we examined ref(2)P levels specifically in the mitochondrial fraction from aged flies. Short-term, midlife Drp1 induction (day 30–37) resulted in reduced ref(2)P levels in the mitochondrial fraction from aged flies (Fig. 6m, n and Supplementary Fig. 6g). Together, these findings suggest that the midlife shift toward more elongated, less circular mitochondria, in flight muscle, contributes to a decline in mitophagy. Short-term Drp1 induction in midlife, for 7 days, promotes mitochondrial fragmentation and facilitates mitophagy. RU486 had no significant impact on mitochondrial DNA levels in aged flight muscle, co-localization of Atg8a/LC3 with mitochondria and accumulation of ref(2)P co-localized with mitochondria (Supplementary Fig. 6b, c, e, f, h and i).

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 Full size image

Midlife Drp1 induction requires autophagy to prolong lifespan

To seek evidence for a causal role for autophagy in Drp1-mediated longevity, we set out to directly manipulate Atg1, a Ser/Thr protein kinase involved in the initiation of autophagosome formation56. More specifically, we inhibited Atg1 by RNAi from midlife onwards in flies with increased Drp1-mediated mitochondrial fission and compared survivorship to flies with increased Drp1-mediated mitochondrial fission alone (Fig. 7a–e). We found that induced RNAi of Atg1 in midlife reduced ATG8a/LC3 levels in aged flight muscle (Fig. 7b, d) and suppressed the lifespan extension associated with midlife Drp1 induction (Fig. 7e, Supplementary Fig. 7a). Inducing RNAi of Atg1 in midlife did not shorten lifespan in control flies (Supplementary Fig. 7b). These results demonstrate that the lifespan-extending effects of promoting Drp1-mediated mitochondrial fission in midlife depend upon the autophagy pathway. In a similar fashion, we find that induced RNAi of Atg1 in midlife suppressed the lifespan extension associated with midlife RNAi of dMfn (Supplementary Fig. 7c).

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 Full size image

Next, we set out to determine whether autophagy is required for Drp1-mediated improvements in mitochondrial function in middle-aged flies. We found that induced RNAi of Atg1 in midlife suppressed the increase in mitochondrial respiratory function, complex I activity and reduced ROS levels associated with midlife Drp1 induction (Fig. 7f–h). Finally, we set out determine whether autophagy was involved in Drp1-mediated improvements in proteostasis in aged animals. Importantly, we found that induced RNAi of Atg1 in midlife suppressed the Drp1-mediated reduction in protein aggregates in aged muscle (Fig. 7i, j). Interestingly, we also observed that promoting Drp1-mediated mitochondrial fission in the context of induced RNAi of Atg1 in midlife leads to increased levels of ubiquitinated proteins in the mitochondrial fraction from aged flies (Fig. 7k, l; Supplementary Fig. 7d).

Taken together, our data indicate that the anti-aging, prolongevity effects of promoting mitochondrial fission, in middle-aged animals, is dependent upon the activity of the autophagy pathway.