Genetic variation in glia–neuron signalling modulates ageing rate

Abstract

The rate of behavioural decline in the ageing population is remarkably variable among individuals. Despite the considerable interest in studying natural variation in ageing rate to identify factors that control healthy ageing, no such factor has yet been found. Here we report a genetic basis for variation in ageing rates in Caenorhabditis elegans. We find that C. elegans isolates show diverse lifespan and age-related declines in virility, pharyngeal pumping, and locomotion. DNA polymorphisms in a novel peptide-coding gene, named regulatory-gene-for-behavioural-ageing-1 (rgba-1), and the neuropeptide receptor gene npr-28 influence the rate of age-related decline of worm mating behaviour; these two genes might have been subjected to recent selective sweeps. Glia-derived RGBA-1 activates NPR-28 signalling, which acts in serotonergic and dopaminergic neurons to accelerate behavioural deterioration. This signalling involves the SIR-2.1-dependent activation of the mitochondrial unfolded protein response, a pathway that modulates ageing. Thus, natural variation in neuropeptide-mediated glia–neuron signalling modulates the rate of ageing in C. elegans.

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Figure 1: Wild strains of C. elegans show varied ageing rates.
Figure 2: Polymorphisms in rgba-1 regulate mating efficiency.
Figure 3: Polymorphisms in npr-28 regulate virility.
Figure 4: RGBA-1–NPR-28 signalling regulates UPRmt via SIR-2.1.
Figure 5: Population genetics of rgba-1 and npr-28 loci.

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Acknowledgements

We thank M.-M. Poo, X. Yu and D. Chen for critical reading of the manuscript; H.-W. Zhu and L.-S. Wang for mass spectrometry analysis; Y. H. Wong and J. Chu for providing the Gα16 and mRuby3 plasmids, respectively; Z. Chen and X. Bai for experimental assistance; and the Caenorhabditis Genetics Center for providing strains. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB 13000000) and the National Natural Science Foundation of China (31471149 and 81527901).

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J.-A.Y. and G.G. performed most of the experiments. X.-J.L. performed male mating, locomotion and male retention assays. Z.-Q.H. and H.-P.L. performed population genetic analysis. K.L. carried out the endoplasmic reticulum fractionation assay. X.-L.K. conducted the male retention assay and helped to determine RGBA-1-2b. H.L., Y.-H.S., and W.-L.H. helped to conduct HPLC and mass spectrometry analysis of neuropeptides. S.-Q.C., J.-A.Y., and G.G. designed the study. S.-Q.C., J.-A.Y., G.G., and H.-P.L. analysed data and wrote the manuscript with input from all authors.

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Correspondence to Shi-Qing Cai.

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Reviewer Information Nature thanks L. Bianchi, P. McGrath and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Cloning of rgba-1.

a, Schematic illustration of generation of SQC0002 strain. CB4856 was crossed with Pbas-1::bas-1::gfp transgenic worms (with N2 genetic background), and about a quarter of aged F2 progeny showed an elevated level of BAS-1::GFP expression. The F2 progeny with high expression levels of BAS-1 were backcrossed with N2 worms eight times; the resulting strain was named SQC0002. b, Left, expression of BAS-1::GFP in SQC0002 and N2 worms at day 9 of adulthood. Scale bar, 10 μm. Representative of n = 5 independent experiments. Right, quantification of BAS-1::GFP fluorescence intensity. GFP fluorescence was normalized to average fluorescence intensity of age-matched N2 worms. c, Mating efficiency of SQC0002, CB4856, and N2 males at a range of ages. d, Whole-genome sequencing of SQC0002 worms identified a 328-kb region in chromosome I that possessed enriched CB4856 alleles. e, Age-dependent changes in mating efficiency in N2, SQC0002, and SQC0002 males with single-copy transgene of empty vector (SQC0002;EV) or rgba-1 (SQC0002;rgba-1). All data shown are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (b, two-sided t test; c, e, ANOVA with Dunnett’s test). For b, c and e, each data point represents the result of one independent experiment. The numbers of tested worms (b) and mating plates (c, e) are shown beneath the bars. Source data

Extended Data Figure 2 CRISPR–Cas9-mediated genome editing in rgba-1 and npr-28 genes.

a, Sequence confirmation of N2;rgba-13V4H, N2;rgba-13G4R, N2;rgba-13V4R, and CB4856;rgba-13G4H worms. The N2;rgba-13V4H, N2;rgba-13G4R, and N2;rgba-13V4R worms were generated by converting the 3G4H rgba-1 allele to 3V4H, 3G4R, and 3V4R, respectively, in N2 worms. The CB4856;rgba-13G4H worms were generated by changing the 3V4R rgba-1 allele to 3G4H in CB4856 worms. Black arrows indicate SNP sites. b, c, Schematic illustrations of molecular details of rgba-1 (b) and npr-28 (c) mutations. Three nucleotides highlighted in purple represent the protospacer-adjacent motif. d, Sequence confirmation of AB3;npr-28N2, AB3;npr-28166L, N2;npr-28AB3, and N2;npr-28166M worms. The AB3;npr-28166L and AB3;npr-28N2 worms were generated by changing the npr-28 allele to the 166L and N2-type npr-28 allele, respectively, in AB3 worms. N2;npr-28166M and N2;npr-28AB3 worms were generated by changing the npr-28 allele to the 166M and the AB3-type npr-28 allele, respectively, in N2 worms. Black arrows indicate SNP sites.

Extended Data Figure 3 Isolation and characterization of RGBA-1-derived peptides.

a, Separation of N2 worm neuropeptides by HPLC. Total neuropeptides were isolated from the mixture of N2 males and hermaphrodites. Grey bar indicates fractions selected for further analysis. mAU, milli-absorbance unit. bf, Tandem mass spectrometry spectrum of RGBA-1-derived peptides. Peptide sequence was confirmed by higher-energy collisional dissociation fragmentation; y-type and b-type ions are shown in the spectrum.

Extended Data Figure 4 Polymorphisms in the signal peptide affect RGBA-1 production.

a, Left, representative images show co-localization of mCherry-fused RGBA-1 signal peptides with an endoplasmic reticulum marker protein calnexin, in HEK293T cells. Scale bar, 10 μm; Right, quantitative analysis of cells with normal, mildly defective, and severely impaired endoplasmic reticulum-localization of RGBA-1 signal peptides. b, Cell fractionation and western blot analyses of mCherry-fused RGBA-1 signal peptides. Cell fractionation was performed by density gradient centrifugation. The endoplasmic reticulum fractions were indicated by the presence of calnexin using anti-calnexin, and mCherry-fused RGBA-1 signal peptides were visualized by anti-mCherry. For gel source data, see Supplementary Fig. 1a. c, Schematic of the RGBA-1::mRuby3 secretion assay. d, e, Images showing RGBA-1-fused mRuby3 fluorescence in glia and coelomocytes (labelled by Punc-122::GFP reporter, ccGFP). The expression of Phsp-16.2::mRuby3 was used as negative control (NC). Arrows point to glial cells and dashed circles indicate coelomocytes. Scale bar, 20 μm. f, Quantitative analysis of the ratio of RGBA-1::mRuby3 fluorescence in coelomocytes to that in glial cells. Each data point represents the result of one independent experiment. The total number of tested worms is shown beneath the bar. Data shown are mean ± s.e.m. ***P < 0.001 (ANOVA with Dunnett’s test). For a and b, n = 4 (a) or 3 (b) independent experiments. Source data

Extended Data Figure 5 Cre–LoxP-mediated recombination and Mos1-mediated single-copy insertion of rgba-1 gene.

a, Schematic representation of Cre–LoxP-mediated recombination of rgba-1 gene. b, The cleavage of rgba-1 in various tissues was verified by PCR. c, Mating efficiency of N2 males at day 1 of adulthood with conditional deletion of rgba-1 in glial cells (rgba-1flox/flox;Pptr-10::Cre or rgba-1flox/flox;Pmir-228::Cre), neurons (rgba-1flox/flox;Prab-3::Cre), or intestinal cells (rgba-1flox/flox;Pges-1::Cre). Data shown are mean ± s.e.m. Each data point represents the result of one independent experiment. d, e, Schematic representations of Mos1-mediated single-copy insertion of rgba-1 (d) or empty vector (e). f, PCR validation of Mos1-mediated insertion of rgba-1. For b and f, n = 2 (b) or 3 (f) independent experiments. For gel source data, see Supplementary Fig. 1b. Source data

Extended Data Figure 6 RNAi screening for BAS-1 regulators identified npr-28.

a, Expression of BAS-1::GFP in N2 worms fed with bacteria that express control or npr-28 double-stranded RNAs. Scale bar, 10 μm. Representative of n = 3 independent experiments. b, Predicted transmembrane topology of NPR-28. The variable residue is highlighted in blue. Alignment of NPR-28 with human somatostatin receptor 5 was used for prediction. c, A phylogenetic tree of NPR-28. d, Expression of NPR-28 in serotonergic, dopaminergic, and motor (or inter-) neurons of N2 males. Serotonergic, dopaminergic, and motor (or inter-) neurons were identified by expression of Ptph-1::mCherry, Pdat-1::mCherry, and Pglr-1::mCherry, respectively. Scale bar, 20 μm. Representative of n = 2 independent experiments. e, Mating efficiency of npr-28-null males selectively expressing N2-type NPR-28 in serotonergic, dopaminergic, and motor (or inter-) neurons at day 1 of adulthood. Two independent transgenic lines per genotype were examined. Data shown are mean ± s.e.m. Each data point represents the result of one independent experiment. f, Alignment of a 30-amino-acid sequence centred at the variable 166th residue from NPR-28 of C. elegans and its Caenorhabditis briggsae, Caenorhadbitis remanei, and Caenorhabditis brenneri homologues. The red asterisk indicates the variation site in wild strains of C. elegans.

Extended Data Figure 7 RGBA-1 and NPR-28 regulate deterioration of multiple behaviours.

a, C. elegans male mating steps. Dashed lines indicate the transport of sperm. b, Number of hermaphrodites contacted by a male before mating initiation. c, d, Turning (c) and vulva location (d) efficiency of males during mating. e, The efficiency of sperm transfer. For be, the numbers of tested males are shown beneath the bars. f, g, Age-dependent changes in pharyngeal pumping rate (f) and locomotion speed (g) in N2, rgba-1, npr-28, and rgba-1;npr-28 mutant worms. The numbers of independent experiments are indicated in parentheses. h, Lifespan curves of N2, rgba-1, npr-28, and rgba-1;npr-28 mutant worms. The numbers of tested hermaphrodites are indicated in parentheses. Data shown in b, d, f and g are mean ± s.e.m., and in c, e, and h represent the sum of animals in three (c, e) or four (h) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (b, d, f, and g, ANOVA with Dunnett’s test; c, e, Fisher’s exact test; h, two-sided log-rank test). Source data

Extended Data Figure 8 The effect of ubl-5 RNAi on UPRmt in rgba-1 and npr-28 worms.

a, Quantification of UPRmt by measuring the fluorescence intensity of Phsp-6::GFP. n = 3 repeated experiments. Each data point represents the result of one independent experiment. Data shown are mean ± s.e.m. *P < 0.05, ***P < 0.001. (ANOVA with Dunnett’s test). b, Fluorescent images of worms expressing the UPRmt reporter Phsp-6::GFP in the presence of ubl-5 RNAi. Representative of n = 3 repeated experiments. Source data

Extended Data Figure 9 UPGMA trees across 249 natural isolates, and global distribution of rgba-1 and npr-28 alleles.

a, UPGMA trees were generated using DNA polymorphisms within a 20-kb region surrounding rgba-1 (left) or npr-28 (right). The minimum basal branch and its descendants are marked in red, and the size of the minimum basal branch nψ = 6 and 5 for rgba-1 and npr-28, respectively. b, Frequency and global distribution of rgba-1 and npr-28 alleles among wild strains. The number of wild strains is indicated inside or near the bar. Source data

Extended Data Table 1 Real-time PCR analysis of transcription of SIR-2.1 downstream genes

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Supplementary Information

This file contains Statistical Test Results, Table and Figure Sample Sizes, Sequences for npr-28 RNAi and ubl-5 RNAi and Supplementary Figure 1, the uncropped blots. (PDF 2586 kb)

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Yin, JA., Gao, G., Liu, XJ. et al. Genetic variation in glia–neuron signalling modulates ageing rate. Nature 551, 198–203 (2017). https://doi.org/10.1038/nature24463

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