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Autophagy protein ATG-16.2 and its WD40 domain mediate the beneficial effects of inhibiting early-acting autophagy genes in C. elegans neurons

Nature Aging volume 4, pages 198–212 (2024)Cite this article

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Abstract

While autophagy genes are required for lifespan of long-lived animals, their tissue-specific roles in aging remain unclear. Here, we inhibited autophagy genes in Caenorhabditis elegans neurons, and found that knockdown of early-acting autophagy genes, except atg-16.2, increased lifespan, and decreased neuronal PolyQ aggregates, independently of autophagosomal degradation. Neurons can secrete protein aggregates via vesicles called exophers. Inhibiting neuronal early-acting autophagy genes, except atg-16.2, increased exopher formation and exopher events extended lifespan, suggesting exophers promote organismal fitness. Lifespan extension, reduction in PolyQ aggregates and increase in exophers were absent in atg-16.2 null mutants, and restored by full-length ATG-16.2 expression in neurons, but not by ATG-16.2 lacking its WD40 domain, which mediates noncanonical functions in mammalian systems. We discovered a neuronal role for C. elegans ATG-16.2 and its WD40 domain in lifespan, proteostasis and exopher biogenesis. Our findings suggest noncanonical functions for select autophagy genes in both exopher formation and in aging.

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Fig. 1: With the exception of atg-16.2, neuronal inhibition of early-acting autophagy genes extends lifespan.
Fig. 2: Neuronal inhibition of all early-acting autophagy genes impairs autophagy.
Fig. 3: Neuronal polyQ aggregation is increased by whole-body inhibition, but reduced by neuronal inhibition of early-acting autophagy genes, except atg-16.2.
Fig. 4: Neuronal inhibition of early-acting autophagy genes, except atg-16.2, induces exophers, which secrete Q40::yfp and extend lifespan.
Fig. 5: atg-16.2 is required for benefits of neuronal inhibition of early-autophagy genes.
Fig. 6: The WD40 domain of ATG-16.2 is dispensable for autophagosome formation but required for exophergenesis.
Fig. 7: The WD40 domain of ATG-16.2 is for benefits of neuronal inhibition of early-autophagy genes.

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Data availability

The authors declare that all data supporting the findings of this study are available within this article, extended data and Supplementary Information. Source data are available with this paper. Additional information on data is available from the corresponding authors upon reasonable request. The unique identifiers for ATG-16.2 sequences are stated in the figure legends.

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Acknowledgements

We thank members of the M.H. and C.K. laboratories for comments on the manuscript. We thank X. She, E. P. Tan, S. Lim and K. Schilling from the M.H. laboratory for creating some of the strains used in this study. We thank H. Zhang for sharing construct atg-16.2p::atg-16.2::gfp, R. Legouis for the GFP::LGG-1(Gly116Ala) strain and B. Grant for sharing the hygromycin resistance-containing plasmid. We thank O. Florey for help with designing the Phe394 mutation in the C. elegans ATG-16.2 WD40 domain. We thank N. Riehs and C. Kenyon for sharing unpublished information on neuronal-only RNAi of autophagy genes. Some of the nematode strains used in this work were provided by the Caenorhabditis Genetics Center (University of Minnesota), which is supported by the NIH Office of Research Infrastructure Program (P40OD010440). We acknowledge the C. elegans Gene Knockout Project at the Oklahoma Medical Research Foundation, which was part of the International C. elegans Gene Knockout Consortium, for generating atg-16.2(ok3224) mutation and WormBase WS283. This work was funded by NIA/NIH fellowship F31AG066405 to M.L.A., a Paul F. Glenn Center Fellowship and Larry L. Hillblom Fellowship 2023-A-019-FEL to H.E., a Taiwan Global Fellowship to L.-H.S., NIH/NIA grants R37AG056510 (to M.D.), R01AG038664 and R01AG039756 and Breakthrough in Gerontology Award BIG 20016 from American Association for Aging Research (to M.H.). The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

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Author notes

  1. Yongzhi Yang

    Present address: Scripps Research Institute, La Jolla, CA, USA

Authors and Affiliations

  1. Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA

    Yongzhi Yang, Caitlin M. Lange, Elizabeth H. Choy, Karie Poon, Tatiana M. Moreno, Anupama Singh, Caroline Kumsta & Malene Hansen

  2. Rutgers, The State University of New Jersey, Nelson Biological Labs, Piscataway, NJ, USA

    Meghan Lee Arnold & Monica Driscoll

  3. Buck Institute for Aging Research, Novato, CA, USA

    Ling-Hsuan Sun, Michael Broussalian, Saam Doroodian, Hiroshi Ebata & Malene Hansen

  4. Leonard Davis School of Gerontology, University of Southern California, Los Angeles, CA, USA

    Ling-Hsuan Sun

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Contributions

Y.Y., M.L.A., C.K. and M.H. designed the experiments. Y.Y., M.L.A., T.M.M., C.K. and M.H. constructed strains. Y.Y., C.M.L., L.-H.S., H.E., M.B., S.D., E.H.C., K.P., T.M.M., A.S., C.K. and M.H. performed lifespan experiments. Y.Y. and C.K. counted polyQ aggregates. Y.Y. and E.H.C. conducted healthspan assays. Y.Y., C.M.L., S.D. and C.K. measured RNAi efficiency. Y.Y. conducted neuronal branching, dye-filling experiments and cloning. C.M.L. and C.K. performed chemotaxis experiments. M.L.A., C.M.L., L.-H.S., H.E. and C.K. monitored exopher formation. L.H.S. monitored exopher formation in polyQ-expressing animals. C.K. performed all autophagy measurements and RT–qPCR. Y.Y., C.K. and M.H. wrote the manuscript with input from all co-authors.

Corresponding authors

Correspondence to Caroline Kumsta or Malene Hansen.

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The authors declare no competing interests. The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Nature Aging thanks Evandro Fang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Neuronal expression of SID-1, an RNA channel protein, leads to RNAi-competent neurons.

(a) Mean GFP fluorescence intensity in head region of rgef-1p::gfp animals on day 1 to day 12 of adulthood, relative to day 1. Error bars are s.d. of n = 3 experiments, with n = 29, 34, 32, 24, 28, 19 animals over 3 independent experiments. ns P = 0.78, P = 0.05, P = 0.116, ****P < 0.0001, **P = 0.005, by one-way ANOVA with Dunnett’s multiple comparisons test. (b-f) Wild-type animals (N2, WT), sid-1; rgef-1p::sid-1 + rgef-1p::gfp, and sid-1 mutants after whole-life RNAi against the indicated gene with tissue-specific functions, compared to control (CTRL). (b) Representative animals are shown with WT animals displaying paralysis (Prz) on unc-112 RNAi, larval arrest (Lva) on rpl-2 RNAi, blister formation (Bli) and larval arrest (Lva) on bli-1 RNAi, and clear (Clr) and larval arrest on elt-2 RNAi. Mean percent phenotypic penetrance after knockdown of genes with functions in (c) body-wall muscle; unc-22 – twitching and uncoordinated movement (Unc) (n = 8 experiments, ****P < 0.0001) and unc-112 – paralysis (n = 7 experiments, ****P < 0.0001), (d) a ubiquitous manner; rpl-2 – larval arrest (n = 8 experiments, ****P < 0.0001), (e) hypodermis; tsp-15 – blisters (n = 8 experiments, ****P < 0.0001) and bli-1 – blisters and larval arrest; (f) intestine; elt-2 – clear and larval arrest. Error bars are s.d. (g) Mean GFP fluorescence intensity in head region of day 1 sid-1; rgef-1p::sid-1 + rgef-1p::gfp rgef-1p::gfp animals after whole-life gfp RNAi compared to control (CTRL). Error bars are s.d. with n = 30 over 3 independent experiments. ****P < 0.0001, **P = 0.005, by two-tailed Student’s t-test. Scale bar: 100 µm. (h) Mean GFP fluorescence intensity in head region of day 1 sid-1; rgef-1p::sid-1 + rgef-1p::gfp rgef-1p::gfp animals after whole-life lgg-1 RNAi (n = 32) compared to control (CTRL) (n = 35). Error bars are s.d. over 3 independent experiments. ****P < 0.0001 by two-tailed Student’s t-test. Scale bar: 100 µm. (i) Mean percent of shrinker phenotype in day 2 WT, rde-1; unc-47p::rde-1::SL2::sid-1 (capable of GABA neuron-specific RNAi), sid-1; rgef-1p::sid-1 + rgef-1p::gfp, or sid-1 animals after two generations of whole-life snb-1 or unc-13 RNAi compared to control (CTRL). Error bars are s.d. with ****P < 0.0001 and ns P > 0.99 by one-way ANOVA with Dunnett’s multiple comparisons test.

Source data

Extended Data Fig. 2 Healthspan and neuronal phenotypes of animals after neuronal inhibition of atg-7 and lgg-1/ATG8.

(a) Mean body bends per 20 s of sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after whole-life atg-7, or lgg-1/ATG8 RNAi compared to control (CTRL). Error bars are s.e.m. of one representative experiment, each with n = 16 animals. Experiment was performed three times with similar results. Linear regression comparison versus CTRL: atg-7 RNAi: Pslope = 0.5; Py-intercept = 0.02; lgg-1/ATG8 RNAi: Pslope = 0.3; Py-intercept = 0.01. (b) Mean number of contractions in the terminal pharyngeal bulb per 30 s of sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after whole-life atg-7, or lgg-1/ATG8 RNAi compared to control (CTRL). Error bars are s.d. of n = 30 animals over 3 independent experiments. Linear regression comparison versus CTRL: atg-7 RNAi: Pslope = 0.4; Py-intercept = 0.4; lgg-1 RNAi: Pslope = 0.3; Py-intercept = 0.5. (c) Mean number of progeny produced per day in sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after atg-7 (n = 19 animals), or lgg-1/ATG8 RNAi (n = 18 animals) compared to control (CTRL) (n = 22 animals) over 2 independent experiments. Error bars are s.d. CTRL versus atg-7 RNAi: ns P = 0.72, 0.89, 0.91, 0.82, >0.99; CTRL versus lgg-1/ATG8 RNAi: ns P = 0.95, 0.96,0.60, 0.70, >0.99, by two-way ANOVA with Dunnett’s multiple comparisons test. (d) Analysis of integrity of sensory neurons in day 5 sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after atg-7, or lgg-1/ATG8 RNAi compared to control (CTRL). Sensory mutants daf-10(e1387) and osm-6(p811) are negative controls. Shown are representative images of n = 10 animals. Experiment was performed three times with similar results. Scale bar, 20 μm. (e) Representative image of neuronal branch (arrowhead) from ALM neuron in day 15 sid-1; rgef-1p::sid-1+ rgef-1p::gfp animals. Scale bar: 20 µm. Mean percent of animals with branches after whole-life atg-7, or lgg-1/ATG8 dsRNA compared to control (CTRL) of n = 4 experiments. Error bars are s.d. ***P = 0.0002, ****P < 0.0001 by Cochran-Mantel-Haenszel test. (f) Mean chemotaxis index of day 5 sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals after whole-life atg-7, or lgg-1/ATG8 RNAi using the chemoattractant butanone. Error bars are 95% C.I. of n = 4 experiments. *P = 0.048, **P = 0.0094, by one-way ANOVA with Dunnett’s multiple comparisons test.

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Extended Data Fig. 3 Autophagy status is unchanged in animals expressing sid-1 and in non-neuronal tissues after neuronal knockdown of early-acting autophagy genes.

(a) Mean neuronal GFP::LGG-1 and GFP::LGG-1(G116A) punctae in day 1 wild-type (sid-1 + /+) (n = 28, 29) and sid-1(qt9) (sid-1-/-) animals (n = 27, 27) with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 26, 29). Error bars are s.d. over 3 independent experiments. Comparison between strains: LGG-1: ns P = 0.75, P = 0.97, G116A: ns P = 0.98, P > 0.99. Comparison of lipidated and unlipidated structures: ****P < 0.0001, by two-way ANOVA with Tukey’s multiple comparisons test. (b) Mean sqst-1p::sqst-1::gfp fluorescence intensity in head region of day 1 wild-type (sid-1 + /+), and sid-1(qt9) (sid-1(-/-)) animals with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) on day 1 of adulthood. Error bars are s.d. of n = 31 animals over 3 independent experiments. ns P = 0.18 and P = 0.59 by one-way ANOVA with Dunnett’s multiple comparisons test. (c) GFP::LGG-1 punctae in intestinal cells of day 1 wild-type (sid-1 + /+) (n = 59) and sid-1(qt9) (sid-1-/-) animals (n = 62) with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 62). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.79, P = 0.99 by one-way ANOVA with Dunnett’s multiple comparisons test. (d) GFP::LGG-1 punctae in body-wall muscle areas of day 1 wild-type (sid-1 + /+) (n = 48) and sid-1(qt9) (sid-1-/-) animals (n = 45) with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 52). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.64, P = 0.70 by one-way ANOVA with Dunnett’s multiple comparisons test. (e) GFP::LGG-1 punctae in intestinal cells of day 1 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; lgg-1p::gfp::lgg-1 animals after whole-life atg-7 (n = 65), or lgg-1/ATG8 (n = 48) RNAi compared to control (CTRL) (n = 81). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.98, P = 0.71 by one-way ANOVA with Dunnett’s multiple comparisons test. (f) GFP::LGG-1 punctae in body-wall muscle areas of day 1 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; lgg-1p::gfp::lgg-1 animals after whole-life atg-7 (n = 54), or lgg-1/ATG8 (n = 53) RNAi compared to control (CTRL) (n = 49). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.34, P = 0.96 by one-way ANOVA with Dunnett’s multiple comparisons test.

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Extended Data Fig. 4 Neuronal PolyQ aggregation, exopher formation and lifespan extension are correlated.

(a) Number of neuronal PolyQ aggregates in day 7 rgef-1::Q40::yfp wild-type (sid-1 + /+) (n = 41) and sid-1(qt9) (sid-1-/-) animals (n = 48 with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 42). Violin plots with solid line indicating median and dashed lines indicating quartiles. ns P = 0.79, P = 0.82 by one-way ANOVA with Dunnett’s multiple comparisons test. (b) Mean percent of ALMR neurons with exophers of day 2 mec-4p::mCherry wild-type (sid-1 + /+) (n = 228 animals) and sid-1(qt9) (sid-1-/-) (n = 262 animals) with or without rgef-1p::sid-1 transgene (rgef-1p::sid-1 (+)) (n = 295 animals). Error bars are s.d. of n = 7 experiments, ns P = 0.62, P = 0.54 by two-sided Cochran-Mantel-Haenszel test. (c) Mean percent of ALMR neurons with exophers of day 2 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; mec-4p::mCherry animals (n = 247 animals) after adult-only atg-7 (n = 271 animals), or lgg-1/ATG8 (n = 285 animals) RNAi compared to control (CTRL). Error bars are s.d. of n = 6 experiments, *P = 0.028, P = 0.00006 by two-sided Cochran-Mantel-Haenszel test. (d) Mean percent of ALMR neurons with exophers of day 2 mec-4p::mCherry animals (n = 151 animals) after whole-life atg-7 (n = 157 animals), or lgg-1/ATG8 (n = 180 animals) RNAi compared to control (CTRL). Error bars are s.d. of n = 5 experiments, ns P = 0.39, P = 0.53 by two-sided Cochran-Mantel-Haenszel test. (e-g) Percent mean lifespan (LS) change (Fig. 1b), number of neuronal PolyQ aggregates (Fig. 3b), and mean percent of AMLR neurons with exophers (Fig. 4b) plotted against each other with simple linear regression (solid line with 95% C.I. as dashed lines). Numbers refer to specific RNAi treatment; 1unc-51/ATG1, 2atg-13, 3bec-1/BECN1, 4atg-9, 5atg-16.2, 6atg-7, 7atg-4.1, 8lgg-1/ATG8, 9cup-5, 10epg-5, 11vha-13, 12vha-15, 13vha-16. P values determined by two-sided Spearman correlation test.

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Extended Data Fig. 5 atg-16.2 and atg-4.1 mutants display similar RNAi phenotypes.

(a-d) Phenotypes of day 2 WT, atg-16.2(ok3224), atg-4.1(bp501), and sid-1(qt9) animals after whole-life RNAi against a specific gene expressed in different tissues. Mean percent phenotypic penetrance after knockdown of genes with functions in (a) body-wall muscle; unc-22 – twitching and uncoordinated movement (Unc) and unc-112 – paralysis, (b) a ubiquitous manner; rpl-2 – larval arrest, (c) hypodermis; tsp-15 – blisters and bli-1 – blisters and larval arrest; (d) intestine; elt-2 – clear and larval arrest. Error bars are s.d. of n = 4 experiments. (a) unc-22 RNAi: ns P = 0.59, P = 0.87, ****P < 0.0001. unc-112 RNAi: ns P = 0.92, P = 0.96, ****P < 0.0001. (b) rpl-2 RNAi: ns P = 0.82, P = 0.96, ****P < 0.0001. (c) tsp-15 RNAi: ns P = 0.99, P = 0.64, ****P < 0.0001. bli-1 RNAi: ns P = 0.24, P = 0.99, ****P < 0.0001. (d) elt-2 RNAi: ns P = 0.76, P = 0.18, ****P < 0.0001 by one-way one-way ANOVA with Dunnett’s multiple comparisons test.

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Extended Data Fig. 6 Multiple sequence alignment of ATG16 proteins, and atg-16.2 mRNA expression levels in C. elegans.

(a) Primary sequences of ATG16 proteins from S. cerevisiae (1 isoform, Sc_ATG16), C. elegans (2 isoforms, Ce_ATG-16.1, Ce_ATG-16.2), Mus musculus (2 isoforms, Mm_ATG16L1, MmATG16L2), and humans (2 isoforms, Hs_ATG16L1, Hs_ATG16L2) were aligned by the Clustal Omega and colored by conservation with darker shades indicated increased conservation (Jalview). Protein elements of ATG16 proteins are drawn under the alignment and phenylalanine 349 and isoleucine 509 are indicated with an arrow. Sequences showed are ATG16 (NP_013882.1) in S. cerevisiae, ATG16.2 (NP_495299.2) in C. elegans, ATG16 (NP_001138124.2) in D. melanogaster, ATG16L1 (NP_001192320.1) in M. musculus, and ATG16L1 (NP_001350671.1) in H. sapiens. (b) Transcript levels of atg-16.2 in wild-type (WT), atg-16.2(ok3224), and atg-16.2(ok3224) animals expressing full-length atg-16.2, atg-16.2(∆WD40), or atg-16.2(Phe394A, Ile509Met) from the neuronal rgef-1 promoter. Schematic of atg-16.2 cDNA indicates the ok3224 deletion, the WD40 domain, the position of the Phe394Ala and Ile509Met point mutations, and the amplicons produced by the primers used in this experiment. Data are the mean and s.e.m. of three biological replicates, each with three technical replicates, and are normalized to the mean expression levels of three housekeeping genes. Amplicon 1: **P = 0.0013, ns P = 0.23, P = 0.36, P = 0.49, Amplicon 2: *P = 0.03, ns P = 0.16, *P = 0.03, ns P = 0.89 by unpaired two-sided t-test with control.

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Supplementary information

Reporting Summary

Supplementary Table 1

Summary of lifespan experiments of comparing WT, sid-1 and neuronal-specific RNAi strains.

Supplementary Table 2

Summary of statistical information for figures testing >10 conditions (Figs. 1–4).

Supplementary Table 3

Summary of lifespan experiments in WT animals after neuronal gene knockdown.

Supplementary Table 4

Summary of lifespan experiments in transgenic strains after neuronal knockdown of atg-7 and lgg-1/ATG8.

Supplementary Table 5

Summary of lifespan experiments of animals clustered ± exopher formation in touch neurons.

Supplementary Table 6

Summary of lifespan experiments in autophagy mutants atg-16.2 and atg-4.1.

Supplementary Table 7

Summary of lifespan experiments with neuronal atg-16.2 reconstitution.

Supplementary Table 8

C. elegans strains used in this study.

Supplementary Table 9

Plasmids used in this study.

Supplementary Table 10

Primers used in this study.

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Yang, Y., Arnold, M.L., Lange, C.M. et al. Autophagy protein ATG-16.2 and its WD40 domain mediate the beneficial effects of inhibiting early-acting autophagy genes in C. elegans neurons. Nat Aging 4, 198–212 (2024). https://doi.org/10.1038/s43587-023-00548-1

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