Circadian autophagy drives iTRF-mediated longevity
Matt Ulgherait, Adil M Midoun, Scarlet J Park, Jared A Gatto, Samantha J Tener, Julia Siewert, Naomi Klickstein, Julie C Canman, William W Ja, Mimi Shirasu-Hiza, Matt Ulgherait, Adil M Midoun, Scarlet J Park, Jared A Gatto, Samantha J Tener, Julia Siewert, Naomi Klickstein, Julie C Canman, William W Ja, Mimi Shirasu-Hiza
Abstract
Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans1-5. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. Here, to exploit the genetic tools and well-characterized ageing markers of Drosophila, we developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. We found that iTRF enhanced circadian-regulated transcription and that iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension.
Conflict of interest statement
Competing Interests:
The authors declare no competing interests.
© 2021. The Author(s), under exclusive licence to Springer Nature Limited.
Figures
Light blue boxes on graphs indicate duration of TRF (aqua), IF (medium blue), or iTRF (sky blue) during lifespan. (a) Schematic of different feeding regimens utilized in Drosophila lifespan screen. (b) 12-hour time-restricted feeding (TRF) from day 10 until death shortened female lifespan (top; ad lib, solid gray, n=292; TRF, dashed gray, n=142) and minimally extended male lifespan (bottom; ad lib, solid, n=241; TRF, dashed, n=314). (c) In contrast, TRF from days 10–40 extended female (top; ad lib, solid gray, n=292; TRF, dashed gray, n=150) and male (bottom; ad lib, solid gray, n=241; TRF, dashed gray, n=406) lifespan. (d) 24-hour intermittent fasting (IF) from day 10-death shortened both female (top; ad lib, solid gray, n=145; IF, dashed gray, n=149) and male (bottom top; ad lib, solid gray, n=241; IF, dashed gray, n=276) lifespan. (e) Intermittent time-restricted feeding (iTRF) from day 10-death did not extend lifespan (ad lib, solid gray, n=142; TRF, dashed gray, n=157). (f) iTRF regimen from days 10–40 extended male lifespan (ad lib, solid gray, n=323; TRF, dashed gray, n=382). (See Methods for trials, statistics, and source data; n=number of individual flies; p-values were obtained by log-rank analysis (b-f).
Light blue boxes on graphs indicate duration of iTRF during lifespan; solid and dashed lines represent flies on ad lib and iTRF diets, respectively. 10-day periods of iTRF in early to mid-life (days 10–40 of adulthood) can extend lifespan but not later in life (days 40–50): (a) days 10–20 with females (ad lib n=311; iTRF n=319); days 20–30 with (b) females (iTRF n=337) and (c) males (ad lib n=323; iTRF n=366); days 30–40 with (d) females (iTRF n=355) and (e) males (iTRF n=293) all extend lifespan. (f, g) iTRF days 40–50 of adulthood did not extend male (iTRF n=302) or female (iTRF n=349) lifespan. (h) Relative to flies on ad lib diet (dark gray dots, n=13), flies on iTRF (shown as blue or green dots depending on diet phase, n=14) starve during the fasting phase (n.a., no food available) and eat more during the feeding phase (green dots). (i) iTRF extended mean lifespan regardless of dietary protein concentration (n=98–347 for each sample of ad lib or iTRF flies at each protein concentration). (j) After partial genetic ablation of insulin producing cells, iTRF still extended lifespan (dashed brown, n=424) relative to ad lib diet (solid brown, n=310), to a similar extent as in genetic controls (ad lib, solid gray, n=161; iTRF, dashed gray, n=180). (See Methods for trials, statistics, and source data; n=number of individual flies; ****=p<0.0001; p-values were obtained by log-rank analysis (a-g, and j) and unpaired two-tailed t-test (h-i). Center values=averages; error bars=SEM.)
(a) Representative western blot of Triton-insoluble protein accumulation of p62/ref(2)P (each sample=30 flies/condition/timepoint; see also SI Figure 1). (b) Quantification of triton insoluble protein levels showed that iTRF flies (light gray) exhibited reduced accumulation of p62/ref(2)P with age, relative to ad lib flies (dark gray) (average of 4 biological repeats). (c) Representative images of 40-day old indirect flight muscle stained for polyubiquitin protein aggregates (green), p62/ref(2)P (magenta), and filamentous actin (F-actin, blue); scale bar=50 µm. iTRF significantly reduced (d) polyubiquitin aggregates and (e) accumulation of p62 aggregates (ad lib n=10 thoraces, iTRF n=11 thoraces). (f) iTRF also reduced age-related intestinal over-proliferation, as marked by phospho-histone H3 staining (p-HH3) (ad lib n=8 guts; iTRF n=9 guts); scale bar=50 µm. (g) Light blue boxes on graphs indicate duration of iTRF during lifespan. iTRF (dashed line) delayed age-related intestinal barrier dysfunction relative to ad lib (solid line), as marked by decreased numbers of smurf flies (n=8–12 cohorts of 20–31 flies). (h-j) Light colored boxes on graphs indicate duration of antibiotic treatment (AB, green) or antibiotic treatment plus iTRF diet (blue/green striped) during lifespan. (h) iTRF flies showed delayed age-related growth in microbiome load with age (n=30 flies/condition/timepoint, 4 biological replicates). iTRF extended lifespan upon microbiome clearance via antibiotics treatment during either (i) total lifespan (ad lib n=227, iTRF n=268) or (j) only days 10–40 of adulthood (ad lib n=144, iTRF n=190). (See Methods for trials, statistics, and source data; n=number of flies unless otherwise indicated; n.s.=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; p-values were obtained by ANOVA followed by Tukey’s post-hoc test (b, g-h), unpaired two-tailed student’s t-test (d-f), and log-rank analysis (i-j). Center values=averages; error bars=SEM.)
(a) Gene expression of timeless, similar to period and Clock, was enhanced by iTRF during the fasting phase (each n=4 biological replicates of 30 female flies/genotype/condition/timepoint; unmarked=n.s.). (b-c) Light blue boxes on graphs indicate duration of iTRF during lifespan. Circadian mutants did not respond to iTRF with extended lifespan relative to controls (ad lib n=187–288; iTRF n=290–311): (b)cycle01 (ad lib n=65, iTRF n=121) and (c)timeless01 (ad lib n=120, iTRF n=152) and period01 (ad lib n=215, iTRF n=184) null mutant females did not respond to iTRF with extended lifespan. (d)cycle01 and (e)period01 mutant females showed a normal “tent-curve” lifespan response to dietary protein titration (n=61–272 flies/genotype/condition/timepoint). (f)period01 mutant females did not starve significantly faster than controls whether they have been on iTRF or ad lib diet (controls: ad lib n=31, iTRF n=35; per: ad lib n=27, iTRF n=42). (g) Similar to controls (gray, ad lib n=30; light gray, iTRF, n=29), period01 mutant females (orange, ad lib n=27; light orange, iTRF, n=27) ate more on iTRF relative to on ad lib diet. (h) Unlike control iTRF flies (light gray), which had reduced accumulation of p62/ref(2)P with age relative to ad lib flies (dark gray), per mutants had similar levels on ad lib (orange) or iTRF (light orange) diets (each dot=1 sample=30 flies; each bar=average of 4 biological repeats). Actin blot is repeated from Figure 2h because the same western blot was used to quantify Ubiquitin, p62/ref(2)P, and actin (loading control); see also SI Figure 1. (i) Representative images of indirect flight muscle from 40-day-old flies stained for filamentous actin (F-actin, blue), ubiquitin (green), and p62/ref(2)P (magenta) showed that, unlike genetic controls (ad lib n=10, iTRF n=11 thoraces), period mutants did not have decreased polyubiquitin, or p62/ref(2)P aggregate accumulation in response to iTRF (ad lib n=10, iTRF n=10 thoraces); scale bar=50 µm. (See Methods for trials, statistics, and source data; n=number of flies unless otherwise indicated; n.s.=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; p-values were obtained by ANOVA followed by Tukey’s post-hoc test (a, g-i) and log-rank analysis (b-c, f). Center values=averages; error bars=SEM.)
(a-b) Similar to circadian genes, iTRF increased the peak amplitude of (a)atg1 and (b)atg8a mRNA expression during the fasting period in wild-type flies (gray) but not period01 mutants (orange) (each dot=1 sample of 30 flies; each bar=average of 4 biological repeats). (c-d)period01 mutants (orange) had (c) reduced activation of AMPK and (d) high levels of TORC1 activity as marked by S6K phosphorylation, both in response to fasting during iTRF, compared to controls (gray) (each dot=1 sample of 30 flies; each bar=average of 4 biological repeats); see also Extended Data Figure 10. (e) Representative images of posterior midgut cells during fasting phase of iTRF of 35-day-old flies labeled with LysoTracker™ (magenta), GFP-Atg8a (green), and DAPI to label the DNA (blue), showed that control animals (n=8 guts; each dot represents 2–3 Z-stacks of the posterior midgut of 1 animal) had high levels of LysoTracker™ and GFP-Atg8a puncta compared to period mutants (n=8 guts); scale bar=20 µm; white dashed boxes on images represent inset area presented in Figure 3g. (See Methods for trials, statistics, and source data; n=number of flies unless otherwise indicated; n.s.=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; p-values were obtained by ANOVA followed by Tukey’s post-hoc test (a-d) and unpaired, two-tailed t-test (e). Center values=averages; error bars=SEM.)
Light blue boxes on graphs indicate duration of iTRF during lifespan. Relative to controls (gray: ad lib, solid, n=161; iTRF, dashed, n=164), which had an ~20% increase in mean lifespan in response to iTRF, circadian overexpression of: (a) dominant-negative (DN) AMPK (K57A, sage green) shortened the lifespan of animals on ad lib diets (solid, n=184) and caused a 13% increase in mean lifespan in response to iTRF (dashed, n=170); (b) constitutively active (CA) AMPK (T184D, dark green) extended lifespan on ad lib diet (solid, n=156) and caused an 8% increase in mean lifespan in response to iTRF (dashed, n=134); (c) dominant-negative (DN) S6K (KQ, light blue) extended lifespan on ad lib diet (solid, n=292) and caused a 12% increase in mean lifespan in response to iTRF (dashed, n=180); (d) constitutively active CA-S6K (STDETE, medium blue) minimally shortened lifespan on ad lib diets (solid, n=237) and caused an 8% increase in mean lifespan in response to iTRF (dashed, n=282). RU486 feeding did not influence control (e) or per01(f) lifespan in flies lacking UAS transgenes (control: ad lib n=136–146, iTRF n=129–142; per01: ad lib n=294–501, iTRF n=238–415). (See Methods for trials, statistics, and source data; n=number of flies; p-values were obtained by log-rank analysis (a-f)).
(a) Using tim-GAL4 to drive expression of mCherry-atg8a, we confirmed oscillating mCherry-Atg8a and free mCherry protein expression by western blot analysis (see also SI Figure 1), which demonstrated circadian autophagic flux in controls on ad lib diet (each lane=30 flies; each time point of quantification=average of 3 biological repeats). (b-d) Solid lines represent ad lib flies; dashed lines represent iTRF flies; light blue boxes on graphs indicate duration of iTRF during lifespan. RNAi-mediated circadian knockdown of (b)atg1 (pink: ad lib n=217, iTRF n=166) and (c)atg8a (purple: ad lib n=196, iTRF n=139) was necessary for iTRF-mediated lifespan extension (controls, gray: ad lib n=194–316, iTRF n=196–409. (d) Circadian overexpression of mCherry-Atg8a was sufficient to extend lifespan on ad lib diet (solid lines: gray, control n=185; purple, mCh-atg8a n=422) and responded minimally to iTRF (dashed lines: gray, control n=421, purple, mCh-atg8a n=437). (See Methods for trials, statistics, and source data; n=number of flies unless otherwise indicated; p-values were obtained by log-rank analysis (b-d). Center values=averages; error bars=SEM.)
(a, c) Light blue boxes on graphs indicate duration of iTRF during lifespan. (a-b) Relative to controls (gray), circadian knockdown of atg1 (pink) increased aging markers of (a) climbing defects (n=10 vials of 10 flies/condition/genotype/timepoint) and (b) protein aggregation (each lane=30 flies; each time point of quantification=average of 4 biological repeats) and made flies resistant to the effects of iTRF (dashed lines, lighter shades), relative to ad lib diets (solid lines, darker shades). (c-d) In contrast, relative to controls (gray), circadian overexpression of atg1 (magenta) decreased aging markers of (c) climbing defects (n=10 vials of 10 flies/condition/genotype/timepoint) and (d) protein aggregation (each lane=30 flies; each time point of quantification=average of 4 biological repeats; see also SI Figure 1) and also made flies resistant to the effects of iTRF (dashed lines), relative to ad lib diets (solid lines). (See Methods for trials, statistics, and source data; n=number of flies unless otherwise indicated; n.s.=p>0.05, ***=p<0.001, ****=p<0.0001; p-values were obtained by ANOVA followed by Tukey’s post-hoc test (a-d). Center values=averages; error bars=SEM.)
(a) RU-induced overexpression of atg1 during iTRF-like phases of the circadian cycle causes circadian enhanced, night-specific expression of atg1 (n=4 biological replicates of 30 flies/timepoint/condition; unmarked=n.s.). (b-c) Light aqua boxes on graphs indicate duration of shifted-TRF during lifespan. (b) Relative to ad lib diet (period01 mutants ad lib, solid orange, n=225), neither night-biased 12:12 treatment of shifted TRF (dashed orange, n=228) or RU-induced atg1 expression (solid magenta, n=319) alone extended the lifespan of per01 mutants. Combined, night-biased 12:12 shifted TRF and RU-induced atg1 expression modestly increased lifespan of per01 mutants (dashed magenta, n=239). (c) Replotted here are per01 mutants on ad lib diet (solid orange, n=225) and on night-biased 12:12 shifted TRF (dashed orange, n=228). Day-biased 12:12 RU-induced exogenous atg1 expression decreased the lifespan of per01 mutants (solid magenta, n=206); this lifespan was increased by night-biased shifted TRF (dashed magenta, n=192). Also shown below are re-plots comparing control and per01 mutant backgrounds with night (b) and day (c) biased RU-induced atg1 expression on ad lib diet (second row) or shifted TRF (third row). (d) Graphic schematic illustrating endogenous rhythms of atg1 expression (gray) and the predicted effects of RU treatment and 12:12 TRF, either night biased and day-biased, on exogenous atg1 expression. (See Methods for trials, statistics, and source data; n=number of flies unless otherwise indicated; n.s.=p>0.05, ***=p<0.001, ****=p<0.0001; p-values were obtained by ANOVA followed by Tukey’s post-hoc test (a) and log-rank analysis (b-c). Center values=averages; error bars=SEM.)
(a) Schematic of ad lib and iTRF diets. (b, d, e) Light blue boxes on graphs=duration of iTRF. (b) Relative to ad lib (solid, n=142), iTRF (dashed, n=205) extended lifespan (p<0.0001). (c) Relative to ad lib (dark gray, n=13), flies on iTRF have increased average food intake (light gray, n=14). (d) iTRF and dietary protein restriction (DR) additively extended lifespan (n>98 for each diet). (e, f) iTRF decreased age-related (e) declines in climbing activity (n=10 vials of 10 flies/genotype/condition/timepoint) and (f) increases in protein aggregation (n=4 biological replicates of 30 flies/genotype/condition/timepoint). (See Methods trials, statistics, and source data; n.s.=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; n=number of flies unless otherwise specified; p-values obtained by unpaired two-tailed t-test (c, f), ANOVA followed by Tukey’s post-hoc test (e), and log-rank analysis (b, d). Center values=averages; error bars=SEM.)
(a) Schematic of the core circadian clock. (b-c) Relative to ad lib diet (solid), iTRF (dashed) enhanced circadian expression of (b)clock and (c)period during fasting (n=4 biological replicates of 30 flies/condition/timepoint; unmarked=n.s.). (d-g) Light blue boxes on graphs=duration of iTRF. (d-e) Relative to ad lib diet (solid), iTRF (dashed) extended the lifespans of controls (gray, ad lib n=145–199, iTRF n=231–262) but not (d)Clkjrk (red-orange, ad lib n=143, iTRF n=196), (e)timCRISPR (gold, ad lib n=228; iTRF n=262 and perCRISPR (orange, ad lib n=179; iTRF n=192) mutants. (f) In contrast to night-biased iTRF (left, dashed gray, n=322), day-biased iTRF (right, dashed gray, n=286) did not extend lifespan relative to ad lib diet (solid gray, n=553); per01 mutants (orange) were not affected by night- or day-biased iTRF (n=209–218). (g-h) Relative to ad lib diet (solid line or dark shade), iTRF (dashed line or light shade) inhibited two aging markers in controls (gray) but not per01 mutants (orange): (g) declines in climbing activity (n=10 vials of 10 flies/genotype/condition/timepoint); and (h) increased protein aggregation (n=4 biological replicates of 30 flies/genotype/condition/timepoint). (See Methods for trials, statistics, and source data; n.s.=p>0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; n=number of flies unless otherwise specified; p-values obtained by ANOVA followed by Tukey’s post-hoc test (b-c, g-h) and log-rank analysis (d-f). Center values=averages; error bars=SEM.)
(a) Schematic of core autophagy mediators. (b-c)atg1 and atg8a expression were circadian-regulated; iTRF (dashed) enhanced night-peaking (b)atg1 and (c)atg8a expression relative to ad lib diet (solid); each n=4 biological replicates of 30 flies/ condition/timepoint (unmarked=n.s.). (d) iTRF (light gray) increased the ratio of free mCherry to mCherry-Atg8a in controls relative to ad lib (dark gray) and, to lesser extent, in per01 mutants (ad lib, orange; iTRF, light orange); each n=4 biological replicates of 30 flies/genotype/condition. (e) LysoTracker™ (magenta), GFP-Atg8a (green), and DNA (blue) staining revealed that control iTRF flies had increased autolysosomes in their intestines (light gray, n=8 guts) compared to per01 mutants (light orange, n=8 guts), scale bar=10 µm. (f-h) Light blue boxes on graphs=duration of iTRF. Relative to ad lib diets (solid), iTRF (dashed) extended the lifespans of controls (gray, ad lib(f) n=195, (g) n=169) iTRF (f) n=263, (g) n=238) but not flies with RNAi-mediated knockdown of (f)atg1 (pink, ad lib n=158, iTRF n=179) or (g)atg8a (purple, ad lib n=151, iTRF n=152) in control backgrounds or (h) in per01 backgrounds (per01mutant, orange, ad lib n=161, iTRF n=167; per01 mutant with atg1-RNAi, hot pink, ad lib n=169, iTRF n=174). (See Methods for trials, statistics, and source data; n=number of flies unless otherwise specified; n.s.=p>0.05, *=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001; p-values obtained by ANOVA followed by Tukey’s post-hoc test (b-d), unpaired two-tailed t-test (e), and log-rank analysis (f-h). Center values= averages; error bars=SEM.)
(a) Schematic of genetic method to knockdown (or overexpress) autophagy components with circadian rhythmicity. (b)per-GAL4 driven atg1 expression (magenta) enhanced circadian, night-peaking atg1 gene expression (gray); each n=4 biological replicates of 30 flies/genotype/timepoint (unmarked=n.s.). (c-d, f-i) Light blue boxes on graphs=duration of iTRF (sky blue) or TRF (aqua) diets. (c-d) Relative to controls (solid gray, n=169–206), genetic circadian overexpression of (c)atg1 (solid magenta, n=139) or (d)atg8a (solid purple, n=191) caused lifespan extension on ad lib diets (solid lines) similar to controls on iTRF (dashed gray, n=139–263) that was not further extended by iTRF (atg1, dashed magenta, n=141; atg8a, dashed purple, n=228). (e) Schematic of pharmacological methods to overexpress atg1 at different times of day. (f) RU486 (RU)-induced atg1 overexpression at night extended lifespan on ad lib diet (solid magenta, n=197) relative to ad lib controls (solid gray, n=186), extending lifespan similar to iTRF controls (dashed gray, n=232) and not further extended by iTRF (dashed magenta, n=329). atg1 overexpression extended lifespan less in (g)per01 mutants (per: ad lib, solid orange, n=192; iTRF, dashed orange, n=245; per with night atg1 OE: ad lib, solid pink, n=225; iTRF, dashed pink, n=345). (h, i) Relative to ad lib controls (solid gray, n=208), shifted-TRF controls (dashed gray, n=363) and 12-hour RU-induced overexpression of atg1 (solid magenta, n=236–290) extended lifespan non-additively (dashed magenta, n=192–341) (h) during the night phase but (i) not during the day phase. (See Methods for trials, statistics, and source data; n=number of flies unless otherwise specified; n.s.=p>0.05, *=p<0.05, ***=p<0.001, ****=p<0.0001; p-values obtained by ANOVA followed by Tukey’s post-hoc test (b), and log-rank analysis (c-d, f-i). Center values=averages; error bars=SEM.)
Source: PubMed