Circadian regulation of mitochondrial uncoupling and lifespan

Abstract

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target.

Fig. 1. Loss of the repressive arm of the transcriptional circadian clock extends male lifespan.

a Schematic of core molecular clock components and circadian transcriptional feedback loop. Relative to controls (gray), mutant males lacking Cyc function (yellow, b) or expressing dominant-negative clock (daGS > UAS-DN-Clock flies fed RU486, orange, c) show reduced lifespan (p < 0.0001 for each circadian mutant vs. control). In contrast, tim⁰¹ mutants (blue, d) and per⁰¹ mutants (green, e) live longer than controls (gray), even on diets with different concentrations of yeast extract (f, g), except for the highest yeast diet (p < 0.001 for each circadian mutant vs. control on each diet). h per⁰¹ mutants with daGS > UAS-DN-S6K fed either RU486 (dashed lines) or vehicle (solid lines) exhibited similar lifespan extension relative to controls containing daGS > UAS-DN-S6K. i per⁰¹ mutant males containing either the dilp2-GAL4 driver alone (solid lines) or dilp2-GAL4 > UAS-reaper and ablated for insulin-producing cells (dashed lines) exhibit similar lifespan extension relative to controls. See Supplementary Table 1 for n and p values for lifespan experiments, particularly multicurve comparisons; p values were obtained by log-rank analysis.

Fig. 2. period mutants exhibit high metabolic rate due to mitochondrial uncoupling.

Relative to controls (gray), per⁰¹ mutants (green) exhibited: a increased feeding rate (n = 6 vials of ten flies/condition, p < 0.01); b decreased survival upon starvation (n ≥ 99 flies per condition, p < 0.001); c lower baseline levels of lipids (left) and increased rate of lipid utilization after 24 h of starvation (right), as shown by quantification of triacylglyceride (TAG) levels (n ≥ 4 samples/condition, 5 flies/sample, both p < 0.0001); d increased respiration, which was reverted by ubiquitous overexpression of Per during adulthood (n = 6 groups of 10 flies per condition); e higher CO₂ production over the circadian day (n = 6 groups of 10 flies/condition and timepoint); f higher respiration rates after 24 h of feeding with rotenone and oligomycin, but not with 2,4-DNP, or stearic acid (n ≥ 5 groups of 10 flies/condition); g increased oxygen consumption rate when stimulated through complexes I, II, and IV relative to controls (n = 4–6 oxygraph runs per condition); h increased leak respiration, using high-resolution respirometry on purified mitochondria (n = 5 oxygraph runs per condition, p < 0.001); i lower membrane potential, measured by JC-1 staining of purified mitochondria (n = 10 mitochondrial preps per condition, p < 0.001); and j faster recovery from cold shock (n = 26–30 flies per condition, p < 0.001). See Supplementary Information for n if not listed here; ^n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; p values were obtained by unpaired two-tailed t-test (a, c, e, g–i), ANOVA followed by Tukey’s post-hoc test (d, f), and log-rank analysis (b, j); error bars represent SEM.

Fig. 3. UCP4C is necessary for period mutant lifespan and sufficient to extend wild-type lifespan.

Relative to controls (gray), per⁰¹ mutants (green) exhibited: a higher expression of Ucp4B and Ucp4C but not Ucp4A; and constitutively high expression of b Ucp4B and c Ucp4C, both of which are circadian-regulated in controls. Relative to controls (gray), per⁰¹ mutants (green) also exhibited the following phenotypes, which were reverted by suppression of Ucp4B/C expression, comparing flies with (dashed lines) or without (solid lines) piggyback mutation of Ucp4B/C: d increased leak respiration by purified mitochondria; e decreased mitochondrial membrane potential; f faster cold shock recovery; and g increased lifespan. Relative to vehicle-fed controls (gray), daGS > UAS-Ucp4C flies fed RU486 to induce constitutive UCP4C overexpression (magenta) exhibited: h higher leak respiration (p < 0.001); i lower mitochondrial membrane potential (p < 0.001); and j increased lifespan (p < 0.0001). k Ubiquitous overexpression of UCP4C in otherwise wild-type flies was sufficient to extend lifespan (gray, dashed) relative to driver-only controls (gray, solid). In per⁰¹ mutants (green, solid), overexpression of UCP4C did not further extend the lifespan of per⁰¹ mutants (green, dashed). See Supplementary Table 1 for n and statistical analysis of lifespans, particularly multicurve comparisons; ^n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001; p values were obtained by unpaired two-tailed t-test (a, h, i), ANOVA followed by Tukey’s post-hoc test (d, e), and log-rank analysis (f, g, j, k); error bars represent SEM.

Fig. 4. Lifespan extension is mediated by loss of Per specifically in the intestine.

Tissue-specific rescue of Per expression (dashed lines) in the per⁰¹ background (green) and controls (gray). a Ubiquitous rescue of Per during adulthood was sufficient to revert per⁰¹ lifespan to control levels. While neuronal overexpression of Per (b) during adulthood did not revert per⁰¹ lifespan, intestinal overexpression of Per (c, d) was sufficient to revert per⁰¹ lifespan to control levels. e Rescue of Per specifically in intestinal stem cells (ISCs) and enteroblasts (EBs) during development or adulthood reverted per⁰¹ lifespan to control levels. f Loss of period through ubiquitous CRISPR-mediated deletion during adulthood extended lifespan of otherwise wild-type flies, with no further lifespan extension in per⁰¹ nulls. CRISPR-mediated deletion of period in g the intestine or h IScs and EBs also extended lifespan. See Supplementary Table 1 for n and p values for lifespan experiments, particularly multicurve comparisons; p values were obtained by log-rank analysis.

Fig. 5. UCP4C in the intestine is necessary for loss of Per-mediated lifespan extension and sufficient to extend wild-type lifespan.

a Whole intestine expression levels of Ucp4C during day and night show oscillations of Ucp4C expression in controls with constitutively high expression in per⁰¹ mutants. b Representative images of the posterior midgut of control (left) and per⁰¹ mutants (right) stained with Hoechst (DNA) and TMRE during the day and night (scale bar = 30 μm). c While wild-type intestines exhibited high membrane potential during the day and low membrane potential at night, per⁰¹ flies exhibited low membrane potential at both times of day. Knockdown of Ucp4C in the whole intestine (d) or ISCs/EBs (e) reverted per⁰¹ lifespan to control levels. Relative to controls (gray), flies overexpressing Ucp4C (magenta) in the whole intestine (f) or ISCs and EBs (g, h) had extended lifespan (p < 0.0001 for each). See Supplementary Table 1 for n and statistical analysis of lifespans; ^n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001; p values were obtained by ANOVA followed by Tukey’s post-hoc test (a, c) and log-rank analysis (d–h); error bars represent SEM.

Fig. 6. Loss of period preserves intestinal homeostasis via increased mitochondrial uncoupling and decreased ROS levels.

a Smurf assays of 45-day-old flies showed per⁰¹ mutants had reduced populations with intestinal barrier dysfunction, a phenotype dependent upon Ucp4C expression, and that overexpression of Ucp4C also reduced intestinal barrier dysfunction. b Quantification of intestinal pHH3+ staining showed that per⁰¹ mutants had lower levels of age-related hyperproliferation dependent on UCP4C expression and that overexpression of UCP4C caused a large reduction in mitotic cells (n = 13–17 intestines). c Representative images of phospho-histone H3 at residue S10 (pHH3) staining of midguts from 45-day flies (scale bar = 40 μm). (d, e) per⁰¹ mutants and Ucp4C-overexpressing flies exhibited delayed esg + cell overproliferation (e) and lower ROS output of all posterior midgut cells, including ISC/EB populations. f Representative images for MitoSOX staining of aged posterior midguts in control esg-GAL4 > GFP flies, per⁰¹; esg-GAL4 > GFP, and esg-GAL4 > GFP;UAS-Ucp4C flies (scale bars = 35 μm (top) and 15 μm (bottom inset location indicated by dashed lines). See Supplementary Table 1 for n and statistical analysis of lifespans; ^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, c, e, f); error bars represent SEM.

Fig. 7. Pharmacological reduction of ROS via uncoupling preserves intestinal homeostasis and extends lifespan.

Flies fed the mitochondrial uncoupler 2,4-DNP (magenta) either throughout their lifespan (a) or only during adulthood (b) showed extended lifespan relative to vehicle controls (gray, p < 0.0001 for each). c 2,4-DNP feeding increased median lifespan in a dose-dependent manner. d Representative images of MitoSOX staining of posterior midguts in esg-GAL4 > GFP flies fed vehicle or DNP (scale bars = 25 μm, top; 10 μm, bottom, inset location indicated by dashed lines). e Flies fed 2,4-DNP exhibited: fewer esg + cells and thus lower ISC/EB overproliferation with age (left); decreased Mitosox staining of the entire posterior midgut and thus decreased mitochondrial superoxide output (middle); and decreased MitoSOX staining specifically in ISCs/EBs marked by GFP (right). f Representative images of midguts from flies exhibiting loss of Notch-mediated tumor formation (scale bar = 35 μm). Relative to vehicle-fed flies, flies with Notch-induced tumor formation that were fed 2,4-DNP exhibited delayed tumor formation as measured by GFP + area in the midgut (g) and extended lifespans (h, p < 0.0001 for each). See Supplementary Table 1 for n and statistical analysis of lifespans; ^n.s.p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; p values were obtained by log-rank analysis (a, b, c, h), unpaired two-tailed t-test (e), and ANOVA followed by Tukey’s post-hoc test (g); error bars represent SEM.