Genetic and Pharmacological Inhibition of Metabotropic Glutamate Receptor Signalling Extends Lifespan in Drosophila

Abstract

Invertebrate models have been instrumental in advancing our understanding of the molecular mechanisms of ageing. The isolation of single gene mutations that both extend lifespan and improve age-related health have identified potential targets for therapeutic intervention to alleviate age-related morbidity. Here, we find that genetic loss of function of the G protein-coupled metabotropic glutamate receptor (DmGluRA) in Drosophila extends the lifespan of female flies. This longevity phenotype was accompanied by lower basal levels of oxidative stress and improved stress tolerance, and differences in early-life behavioural markers. Gene expression changes in DmGluRA mutants identified reduced ribosome biogenesis, a hallmark of longevity, as a key process altered in these animals. We further show that the pro-longevity effects of reduced DmGluRA signalling are dependent on the fly homologue of Fragile X Mental Retardation Protein (FMRP), an important regulator of ribosomal protein translation. Importantly, we can recapitulate lifespan extension using a specific pharmacological inhibitor of mGluR activity. Hence, our study identifies metabotropic glutamate receptors as potential targets for age-related therapeutics.

Keywords: Drosophila; ageing; metabotropic glutamate receptor; ribosome biogenesis.

FIGURE 1

Mutation of the metabotropic glutamate receptor (DmGluRA) in Drosophila extends female lifespan. (A) Survival of DmGluRA ^[112b] homozygous mutants compared to w ^Dah controls (w ^Dah males: n = 127 dead/8 censored; DmGluRA ^[112b] males: n = 117 dead/18 censored; p = 0.8, Log‐rank test. w ^Dah females: n = 136 dead/6 censored; DmGluRA ^[112b] females: n = 117 dead/19 censored; p = 4.6 × 10⁻²³, Log‐rank test). (B) Number of eggs laid per fly over 24‐h for 10‐day‐old females. Box‐and‐whisker plot represents minimum, 25%, median, 75% and maximum. Individual data points represent average number of eggs laid per fly per vial (15 flies per vial, n = 10 vials). No significant difference: unpaired t‐test, p = 0.51. (C) Survival of virgin female DmGluRA ^[112b] homozygous mutants compared to virgin female w ^Dah controls (w ^Dah virgin females: n = 133 dead/19 censored; DmGluRA ^[112b] virgin females: n = 173 dead/14 censored; p = 1.4 × 10⁻²⁵, Log‐rank test).

FIGURE 2

DmGluRA transcripts show sex‐ and age‐dependent effects on expression. Relative DmGluRA transcript levels measured by qRT‐PCR for total RNA extracted from (A) whole flies (n = 5 biologically independent samples, effect of age p = 0.00039, sex p < 10⁻⁴, sex‐by‐age interaction p = 0.02975, LM); (B) isolated heads (n = 5 biologically independent samples, effect of age p < 10⁻⁴, sex p = 0.001, sex‐by‐age interaction not significant, LM); (C) isolated thoraces (n = 5 biologically independent samples, effect of age p < 10⁻⁴, sex p = 0.00005, sex‐by‐age interaction not significant, LM), and (D) isolated abdomens (n = 5 biologically independent samples, effect of age and sex not significant, sex‐by‐age interaction p = 0.036, LM). Box‐and‐whisker plots represent minimum, 25%, median, 75% and maximum with values for individual biological replicates overlaid as points.

FIGURE 3

DmGluRA mutants are resistant to starvation and oxidative stress. (A) Survival of DmGluRA ^[112b] homozygous mutants compared to w ^Dah controls during starvation. Males (black lines): w ^Dah n = 146 dead/0 censored, DmGluRA ^[112b] n = 146 dead/0 censored; p = 0.73, Log‐rank test; females (red lines): w ^Dah n = 148 dead/0 censored, DmGluRA ^[112b] n = 150 dead/0 censored; p = 2.4 × 10⁻⁵, Log‐rank test. (B) Survival of DmGluRA ^[112b] homozygous mutants compared to w ^Dah controls in the presence of 20 mM paraquat. Males (black lines): w ^Dah n = 147 dead/0 censored, DmGluRA ^[112b] n = 147 dead/0 censored; p = 5.1 × 10⁻⁵, Log‐rank test; females: w ^Dah n = 150 dead/0 censored; DmGluRA ^[112b] n = 148 dead/0 censored; p = 4.2 × 10⁻⁵, Log‐rank test. (C) Representative Western blots and quantification of GST::GFP expression in 10‐day‐old male and female w ^Dah controls and DmGluRA ^[112b] homozygous mutants normalised to actin. Box‐and‐whisker plots represent minimum, 25%, median, 75% and maximum with values for individual biological replicates overlaid as points (n = 6 biologically independent samples, males p = 0.038, females p = 0.018, t‐test).

FIGURE 4

Loss of DmGluRA impacts on neuromuscular health. (A) Climbing/negative geotaxis assay. Each point shows the mean height climbed ± SEM during negative geotaxis assays for w ^Dah control and DmGluRA ^[112b] homozygous females. At the start of the experiment (week 1) n = 68–69 flies. As the experiment progressed flies began to die so at week 8, n = 13–39. No significant effect of genotype, age p < 2 × 10⁻¹⁶, genotype‐by‐age interaction p = 0.0002, LM. (B–D) Exploratory walking duration, velocity and walking distance of w ^Dah control and DmGluRA ^[112b] homozygous females. Each point shows mean ± SEM (n = 23–28, two batches). There was a significant effect of genotype (p < 0.001), age (p < 0.001) and a significant genotype‐by‐age interaction (p < 0.01) for all three measured parameters, mixed effects linear model (with batch as a random effect). **Post hoc t‐test.

FIGURE 5

Loss of DmGluRA activity is associated with changes in ribosomal RNA production and processing. (A) Volcano plot of differential gene expression in DmGluRA ^[112b] mutants versus w ^Dah controls (FDR 0.05%) shown in red. Nine hundred and eighty‐three genes show upregulated expression and 1288 genes show downregulated expression. Each dot represents an individual gene. Horizontal black dotted line denotes adjusted p‐value threshold of −log₁₀(0.05). (B) Gene ontology (GO) analysis of the differentially expressed genes in DmGluRA ^[112b] mutants. Bubble plot shows the top GO terms (FDR < 0.05) for a ranked list of all differentially expressed genes. (C) Representative images of nucleoli (Fibrillarin immunostaining) in the midgut enterocytes of w ^Dah and DmGluRA ^[112b] homozygous mutant flies (scale bar, 10 μm) and quantification of nucleolar size in midgut enterocyte nuclei (determined as each nucleolar area divided by each nuclear area). Box‐and‐whisker plots represent minimum, 25%, median, 75% and maximum with values for biological replicates overlaid as points (average nucleolar area/nuclear area of 10 cells per fly, n = 5 flies, p = 0.0019, t‐test). (D) Ratio of RNA to DNA for the sequences present in pre‐rRNA‐rDNA (n = 8 biologically independent samples, effect of genotype p = 0.0199, but no significant effect of the target sequence or interaction, LM). (E) Representative Western blot and quantification of puromycin incorporation normalised to tubulin. Box‐and‐whisker plots represent minimum, 25%, median, 75% and maximum overlaid with individual data points (n = 5 individual flies, p = 0.0433, t‐test).

FIGURE 6

Pro‐longevity effects of reduced DmGluRA signalling are dependent on FMRP. (A) Representative Western blot and quantification of FMRP expression in 10‐day‐old female w ^Dah and DmGluRA ^[112b] homozygous mutant flies normalised to actin. Box‐and‐whisker plots represent minimum, 25%, median, 75% and maximum with data points for individual biological replicates overlaid (n = 5 biologically independent samples, p = 0.0024, t‐test). (B) Survival of female flies either homozygous mutant for DmGluRA ^[112b] alone or combined with the Fmr ^[392] allele. w ^Dah (n = 129 dead/4 censored), DmGluRA ^[112b] homozygotes (n = 137 dead/0 censored), Fmr ^[392] homozygotes (n = 117 dead/3 censored) and Fmr ^[392] ;DmGluRA ^[112b] double mutants (n = 152 dead/2 censored; Fmr ^[392] vs. Fmr ^[392] ;DmGluRA ^[112b] , p = 2.798 × 10⁻¹⁷, Log‐rank test). (C) Quantification of nucleolar size in midgut enterocyte nuclei (determined as each nucleolar area divided by each nuclear area). Box‐and‐whisker plots represent minimum, 25%, median, 75% and maximum with values for biological replicates overlaid as points (average nucleolar area/nuclear area of 10 cells per fly, n = 5 flies, p < 0.0001, ANOVA). (D) Survival of w ^Dah female flies in the presence of DMSO alone (0 μM) or 10, 50 or 200 μM 2‐Methyl‐6‐(phenylethynyl)pyridine (MPEP), a potent pharmacological inhibitor of mGluRA (0 μM MPEP: n = 134 dead/11 censored; 10 μM MPEP: n = 143 dead/7 censored; 50 μM MPEP: n = 137 dead/11 censored; 200 μM MPEP: n = 138 dead/11 censored; 0 μM vs. 50 μM, p = 0.032 Log‐rank test; 0 μM vs. 200 μM, p = 1.36 × 10⁻⁵, Log‐rank test).