Independent glial subtypes delay development and extend healthy lifespan upon reduced insulin-PI3K signalling

Abstract

Background: The increasing age of global populations highlights the urgent need to understand the biological underpinnings of ageing. To this end, inhibition of the insulin/insulin-like signalling (IIS) pathway can extend healthy lifespan in diverse animal species, but with trade-offs including delayed development. It is possible that distinct cell types underlie effects on development and ageing; cell-type-specific strategies could therefore potentially avoid negative trade-offs when targeting diseases of ageing, including prevalent neurodegenerative diseases. The highly conserved diversity of neuronal and non-neuronal (glial) cell types in the Drosophila nervous system makes it an attractive system to address this possibility. We have thus investigated whether IIS in distinct glial cell populations differentially modulates development and lifespan in Drosophila.

Results: We report here that glia-specific IIS inhibition, using several genetic means, delays development while extending healthy lifespan. The effects on lifespan can be recapitulated by adult-onset IIS inhibition, whereas developmental IIS inhibition is dispensable for modulation of lifespan. Notably, the effects we observe on both lifespan and development act through the PI3K branch of the IIS pathway and are dependent on the transcription factor FOXO. Finally, IIS inhibition in several glial subtypes can delay development without extending lifespan, whereas the same manipulations in astrocyte-like glia alone are sufficient to extend lifespan without altering developmental timing.

Conclusions: These findings reveal a role for distinct glial subpopulations in the organism-wide modulation of development and lifespan, with IIS in astrocyte-like glia contributing to lifespan modulation but not to developmental timing. Our results enable a more complete picture of the cell-type-specific effects of the IIS network, a pathway whose evolutionary conservation in humans make it tractable for therapeutic interventions. Our findings therefore underscore the necessity for cell-type-specific strategies to optimise interventions for the diseases of ageing.

Keywords: Ageing; Astrocytes; Glia; Insulin signalling; Lifespan.

Fig. 1

Glial Pten over-expression extends lifespan and delays development. a qPCR from head RNA shows over-expression of Pten in 1-week-old w^Dah;UAS-Pten/+;repo-GAL4/+ flies (red) compared to control w^Dah;+;repo-GAL4/+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies. n = 4 replicates of 12 heads per replicate per genotype. b Survival curves show extended lifespan for w^Dah;UAS-Pten/+;repo-GAL4/+ flies (red) compared to control w^Dah;+;repo-GAL4/+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies. n > 150 deaths counted per group. c The number of eggs laid by female flies of the indicated ages was not affected by genotype. n = 15 vials of 12 flies per vial per genotype. d The weight of 1-week-old female flies was not affected by genotype. n = 14 flies per genotype. e Egg-to-adult timing shows delayed development for w^Dah;UAS-Pten/+;repo-GAL4/+ flies (red) compared to control w^Dah;+;repo-GAL4/+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies. n > 200 flies counted per group. f Confocal z-projections show glial nuclei immunostained for REPO (magenta) in the central brain. Dotted lines indicate traces of central brain areas. g Central brain areas were unchanged in 1-week-old w^Dah;UAS-Pten/+;repo-GAL4/+ flies (red) compared to control w^Dah;+;repo-GAL4/+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies. n = 7–8 brains per group. h Confocal z-projections show glial nuclei immunostained for REPO (magenta) and glial membranes labelled with mCD8::GFP (green). Dotted lines indicate areas imaged at higher magnification below. i, j REPO-positive glial nuclei were counted and the extent of glia measured by GFP-positive area in the layer of glia shown in h. Both the density of glial nuclei and the extent of glial membranes were unaffected in 1-week-old w^Dah;UAS-Pten/+;repo-GAL4/UAS-mCD8::GFP flies (red) compared to control w^Dah;+;repo-GAL4/UAS-mCD8::GFP (blue) flies. n = 7 brains per group. p values are from Tukey’s multiple comparisons (a), log-rank test versus control group of that colour (b, e), 2-way ANOVA (c), 1-way ANOVA (d, g), or unpaired t-test (i, j)

Fig. 2

Over-expression of wild-type Pten, but not lipid phosphatase dead Pten, extends lifespan and delays development. a, b Survival curves show a extended lifespan for w^Dah;Actin-GeneSwitch/UAS-Pten;+ flies and b unchanged lifespan for w^Dah;Actin-GeneSwitch/UAS-Pten-G137E;+ flies reared on food containing 200 μM RU-486 (induced, red and pink curves) from 2 days of age compared to sibling flies reared on vehicle control food (uninduced, black and grey curves). c, d Survival curves show c extended lifespan for w^Dah;UAS-Pten/+;repo-GAL4/+ flies (red) compared to control w^Dah;+;repo-GAL4/+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies, and d unchanged lifespan for w^Dah;UAS-Pten-G137E/+;repo-GAL4+ flies (pink) compared to control w^Dah;+;repo-GAL4/+ (blue) and w^Dah;UAS-Pten-G137E/+;+ (grey) flies. For all lifespans, n > 130 deaths counted per group. e, f Egg-to-adult timing shows e delayed development for w^Dah;UAS-Pten/+;repo-GAL4/+ flies (red) compared to control w^Dah;+;repo-GAL4/+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies, and f no delay in development for w^Dah;UAS-Pten-G137E/+;repo-GAL4/+ flies (pink) compared to control w^Dah;+;repo-GAL4/+ (blue) and w^Dah;UAS-Pten-G137E/+;+ (grey) flies. n > 55 flies per group. Note that the repo-GAL4/+ curves in c, d and e, f are identical, as these experiments were run in parallel. g, h qPCR from head RNA shows g over-expression of Pten in both w^Dah;UAS-Pten/+;repo-GAL4/+ and w^Dah;UAS-Pten-G137E/+;repo-GAL4/+ flies using primers outside of the G137 region and h over-expression of wild-type Pten in only w^Dah;UAS-Pten/+;repo-GAL4/+ flies using primers designed to amplify the wild-type sequence for G137. n = 4 replicates of 10–12 heads per replicate. p values are from log-rank tests (a–f) or Bonferroni’s multiple comparison (g, h) tests versus control group of that colour

Fig. 3

Adult-specific IIS inhibition in glia extends lifespan in a foxo-dependent manner. a Confocal z-projections show adult (1-week-old) brains with glial nuclei immunostained for REPO (magenta) and glial membranes labelled with mCD8::GFP (green) in 1-week-old w^Dah;+;GSG3285-1/UAS-mCD8::GFP flies reared on food containing 200 μM RU-486 or vehicle control from 2 days of age. b qPCR from head RNA shows over-expression of Pten in 2-week-old w^Dah;UAS-Pten/+;GSG3285-1/+ flies reared on food containing 200 μM RU-486 (induced, red) or vehicle control (uninduced, black) from 2 days of age. n = 6 replicates of 12 heads per replicate per genotype. c–f Survival curves show extended lifespan for (c, d) w^Dah;UAS-Pten/+;GSG3285-1/+, e w^Dah;UAS-InR^DN/+;GSG3285-1/+, and f w^Dah;UAS-dp110^DN/+;GSG3285-1/+ flies reared on food containing 200 μM RU-486 (red curves) from 2 days of age compared to vehicle control (black curves). However, d w^Dah;UAS-Pten/+;GSG3285-1/+ flies reared on food containing 200 μM RU-486 during larval development alone (blue curve) showed no significant change in lifespan, and flies reared on RU-486 throughout both larval development and adulthood (orange curve) showed no additive lifespan extension from adult-only induction. g Survival curves show extended lifespan for w^Dah;UAS-Pten/+;GSG3285-1/+ flies (induced, red curve, and uninduced, black curve) and unchanged lifespan for w^Dah;UAS-Pten/+;GSG3285-1,foxo∆/foxo∆ flies (induced, orange curve, and uninduced, blue curve) reared on food containing 200 μM RU-486 from 2 days of age. h Egg-to-adult timing shows delayed development for w^Dah;UAS-Pten/+;GSG3285-1/+ larvae (induced, red curve; and uninduced, black curve), and a lesser delay of development for w^Dah;UAS-Pten/+;GSG3285-1,foxo∆/foxo∆ larvae (induced, orange curve; and uninduced, blue curve), reared on food containing 200 μM RU-486. For all lifespans, n > 140 deaths counted per group; for development assays, n > 40 flies counted per group. p values are from unpaired t test (a), log-rank test versus uninduced (c, e, f), log-rank test for group of that colour versus uninduced (d), or Cox proportional hazards (g, h)

Fig. 4

Pten over-expression in astrocyte-like glia is sufficient to extend lifespan without delaying development. a Drosophila glia comprise several distinct subsets, including the perineurial and subperineurial glia that form the blood-brain barrier, cortex glia that surround neuronal cell bodies, ensheathing glia that line the neuropil, and astrocyte-like glia whose processes infiltrate the neuropil. Panel adapted from [40]. b Survival curves show reduced lifespan for w^Dah;UAS-Pten/NP6293-GAL4;+ flies (red) compared to control w^Dah;NP6293-GAL4/+;+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies. c Survival curves show reduced lifespan for w^Dah;UAS-Pten/NP2222-GAL4;+ flies (red) compared to control w^Dah;NP2222-GAL4/+;+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies. d Survival curves show extended lifespan for w^Dah;UAS-Pten/+;alrm-GAL4/+ flies (red) compared to control w^Dah;+;alrm-GAL4/+ (blue) and w^Dah;UAS-Pten/+;+ (black) flies. For all lifespans, n > 140 deaths counted per group. e Egg-to-adult timing shows unaffected development timing for w^Dah;UAS-Pten/+;+ flies (black) compared to control wDah;+;+ flies (green). f Egg-to-adult timing shows delayed development timing for w^Dah;UAS-Pten/NP6293-GAL4;+ flies (red) compared to control w^Dah;NP6293-GAL4/+;+ flies (blue). g Egg-to-adult timing shows delayed development timing for w^Dah;UAS-Pten/NP2222-GAL4;+ flies (red) compared to control w^Dah;NP2222-GAL4/+;+ flies (blue). h Egg-to-adult timing shows unaffected development timing for w^Dah;UAS-Pten/+;alrm-GAL4/+ flies (red) compared to control w^Dah;+;alrm-GAL4/+ flies (blue). For all development assays, n > 70 flies counted per group. p values are from log-rank tests versus control group of that colour