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a, Representative images of nerve-ring neurons and mean number of GFP::LGG-1/ATG8 punctae of day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1 animals after <t>whole-life</t> <t>autophagy</t> gene RNAi. Error bars are s.d. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test. See Supplementary Table 2 for n, all P values and statistical details. The brightness of the image displaying lgg-1/ATG8 RNAi was artificially enhanced. Scale bar, 10 μm. b, Representative images of nerve-ring neurons and mean number of non-lipidated GFP::LGG-1/ATG8(Gly116Ala) punctae in day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1(Gly116Ala) animals after whole-life autophagy gene RNAi. Error bars are s.d. P values as in a. See Supplementary Table 2 for all P values and statistical details. The brightness of the nerve-ring image after lgg-1/ATG8 RNAi is artificially enhanced. Scale bar, 10 μm. c, Mean number of GFP::LGG-1/ATG8 punctae in nerve-ring neurons in day 1 rgef-1p::gfp::lgg-1 (WT)-expressing animals after injection of animals with vehicle (dimethylsulfoxide (DMSO); n = 27 animals) or <t>BafA</t> (n = 34 animals), and in animals expressing rgef-1p::gfp::lgg-1(Gly116Ala) (Gly116Ala; DMSO, n = 23; BafA, n = 28 animals), over three independent experiments. Error bars are s.d. NS P = 0.99, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparisons test. d, Mean number of GFP::LGG-1/ATG8 punctae in nerve-ring neurons after injection of animals with vehicle (DMSO) or BafA in day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1 animals after whole-life atg-7 RNAi (DMSO, n = 30; BafA, n = 34 animals, NS P = 0.051) or lgg-1/ATG8 RNAi (DMSO, n = 33; BafA, n = 38 animals, NS P > 0.99) compared to control (CTRL) (DMSO, n = 27; BafA, n = 34 animals; ****P < 0.0001) over three independent experiments. Error bars are s.d. CTRL-DMSO versus atg-7-DMSO: NS P = 0.12; CTRL-DMSO versus lgg-1-DMSO: ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparisons test. e, Representative images and mean fluorescence intensity in head regions of day 1 sid-1; rgef-1p::sid-1; sqst-1p::sqst-1::gfp animals after whole-life autophagy gene RNAi. Error bars are s.d. P values as in a. See Supplementary Table 2 for n, all P values and statistical details. Scale bar, 100 μm. Shading of atg-16.2 emphasizes this RNAi treatment as an exception for lifespan extension by early-acting autophagy genes (Fig. 1).

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

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

Journal: Nature aging

doi: 10.1038/s43587-023-00548-1

a, Representative images of nerve-ring neurons and mean number of GFP::LGG-1/ATG8 punctae of day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1 animals after whole-life autophagy gene RNAi. Error bars are s.d. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test. See Supplementary Table 2 for n, all P values and statistical details. The brightness of the image displaying lgg-1/ATG8 RNAi was artificially enhanced. Scale bar, 10 μm. b, Representative images of nerve-ring neurons and mean number of non-lipidated GFP::LGG-1/ATG8(Gly116Ala) punctae in day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1(Gly116Ala) animals after whole-life autophagy gene RNAi. Error bars are s.d. P values as in a. See Supplementary Table 2 for all P values and statistical details. The brightness of the nerve-ring image after lgg-1/ATG8 RNAi is artificially enhanced. Scale bar, 10 μm. c, Mean number of GFP::LGG-1/ATG8 punctae in nerve-ring neurons in day 1 rgef-1p::gfp::lgg-1 (WT)-expressing animals after injection of animals with vehicle (dimethylsulfoxide (DMSO); n = 27 animals) or BafA (n = 34 animals), and in animals expressing rgef-1p::gfp::lgg-1(Gly116Ala) (Gly116Ala; DMSO, n = 23; BafA, n = 28 animals), over three independent experiments. Error bars are s.d. NS P = 0.99, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparisons test. d, Mean number of GFP::LGG-1/ATG8 punctae in nerve-ring neurons after injection of animals with vehicle (DMSO) or BafA in day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1 animals after whole-life atg-7 RNAi (DMSO, n = 30; BafA, n = 34 animals, NS P = 0.051) or lgg-1/ATG8 RNAi (DMSO, n = 33; BafA, n = 38 animals, NS P > 0.99) compared to control (CTRL) (DMSO, n = 27; BafA, n = 34 animals; ****P < 0.0001) over three independent experiments. Error bars are s.d. CTRL-DMSO versus atg-7-DMSO: NS P = 0.12; CTRL-DMSO versus lgg-1-DMSO: ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparisons test. e, Representative images and mean fluorescence intensity in head regions of day 1 sid-1; rgef-1p::sid-1; sqst-1p::sqst-1::gfp animals after whole-life autophagy gene RNAi. Error bars are s.d. P values as in a. See Supplementary Table 2 for n, all P values and statistical details. Scale bar, 100 μm. Shading of atg-16.2 emphasizes this RNAi treatment as an exception for lifespan extension by early-acting autophagy genes (Fig. 1).

Figure Legend Snippet: a, Representative images of nerve-ring neurons and mean number of GFP::LGG-1/ATG8 punctae of day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1 animals after whole-life autophagy gene RNAi. Error bars are s.d. NS P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by one-way analysis of variance (ANOVA) with Dunnett’s multiple-comparison test. See Supplementary Table 2 for n, all P values and statistical details. The brightness of the image displaying lgg-1/ATG8 RNAi was artificially enhanced. Scale bar, 10 μm. b, Representative images of nerve-ring neurons and mean number of non-lipidated GFP::LGG-1/ATG8(Gly116Ala) punctae in day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1(Gly116Ala) animals after whole-life autophagy gene RNAi. Error bars are s.d. P values as in a. See Supplementary Table 2 for all P values and statistical details. The brightness of the nerve-ring image after lgg-1/ATG8 RNAi is artificially enhanced. Scale bar, 10 μm. c, Mean number of GFP::LGG-1/ATG8 punctae in nerve-ring neurons in day 1 rgef-1p::gfp::lgg-1 (WT)-expressing animals after injection of animals with vehicle (dimethylsulfoxide (DMSO); n = 27 animals) or BafA (n = 34 animals), and in animals expressing rgef-1p::gfp::lgg-1(Gly116Ala) (Gly116Ala; DMSO, n = 23; BafA, n = 28 animals), over three independent experiments. Error bars are s.d. NS P = 0.99, ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparisons test. d, Mean number of GFP::LGG-1/ATG8 punctae in nerve-ring neurons after injection of animals with vehicle (DMSO) or BafA in day 1 sid-1; rgef-1p::sid-1; rgef-1::gfp::lgg-1 animals after whole-life atg-7 RNAi (DMSO, n = 30; BafA, n = 34 animals, NS P = 0.051) or lgg-1/ATG8 RNAi (DMSO, n = 33; BafA, n = 38 animals, NS P > 0.99) compared to control (CTRL) (DMSO, n = 27; BafA, n = 34 animals; ****P < 0.0001) over three independent experiments. Error bars are s.d. CTRL-DMSO versus atg-7-DMSO: NS P = 0.12; CTRL-DMSO versus lgg-1-DMSO: ****P < 0.0001, by two-way ANOVA with Tukey’s multiple-comparisons test. e, Representative images and mean fluorescence intensity in head regions of day 1 sid-1; rgef-1p::sid-1; sqst-1p::sqst-1::gfp animals after whole-life autophagy gene RNAi. Error bars are s.d. P values as in a. See Supplementary Table 2 for n, all P values and statistical details. Scale bar, 100 μm. Shading of atg-16.2 emphasizes this RNAi treatment as an exception for lifespan extension by early-acting autophagy genes (Fig. 1).

Techniques Used: Comparison, Expressing, Injection, Control, Fluorescence

a, Mean percentage of ALMR neurons with exophers in day 2 WT, atg-16.2(ok3224) and atg-4.1(bp501) animals expressing mec-4p::mCherry. Error bars are the s.d. WT (n = 261 animals) versus atg-16.2 (n = 274 animals) (n = 7 experiments, **P = 0.007), WT (n = 273 animals) versus atg-4.1 (n = 243 animals) (n = 6 experiments, ****P < 0.0001) by two-sided Cochran–Mantel–Haenszel test. b, Mean GFP fluorescence intensity in head region in day 1 WT, atg-4.1(bp501) and atg-16.2(ok3224) animals expressing sqst-1p::sqst-1::gfp. Error bars are the s.d. WT (n = 30) versus atg-16.2 (n = 35), ****P < 0.0001; WT (n = 38) versus atg-4.1 (n = 58), ****P < 0.0001) by two-sided t-test over three independent experiments. Representative images from one experiment. Scale bar, 200 μm. c, Mean neuronal GFP::LGG-1/ATG8-positive and GFP::LGG-1(Gly116Ala)-positive punctae in day 1 WT, atg-4.1(bp501) and atg-16.2(ok3224) animals. Error bars are the s.d. WT-GFP::LGG-1 (n = 57) versus WT-GFP::LGG-1(Gly116Ala) (n = 61), ****P < 0.0001; atg-16.2-GFP::LGG-1 (n = 31) versus atg-16.2-GFP::LGG-1(Gly116Ala) (n = 32), ****P < 0.0001; atg-4.1-GFP::LGG-1 (n = 30) versus atg-4.1-GFP::LGG-1(Gly116Ala) (n = 28), NS P = 0.15. Comparison between strains: WT (n = 27) versus atg-16.2 (n = 31), ****P < 0.0001, WT (n = 30) versus atg-4.1 (n = 30) NS P = 0.75 > 0.05, ****P < 0.0001, over three independent experiments by two-way ANOVA with Tukey’s multiple-comparisons test. d, Mean neuronal GFP::LGG-1/ATG8-positive punctae in day 1 WT, atg-4.1(bp501) and atg-16.2(ok3224) animals after vehicle (DMSO) or BafA injections to block autophagy. Error bars are the s.d. WT-DMSO (n = 27) versus WT-BafA (n = 34), ****P < 0.0001; atg-16.2-DMSO (n = 28) versus atg-16.2-BafA (n = 34), *P = 0.043; atg-4.1-DMSO (n = 24) versus atg-4.1-BafA (n = 31), **P = 0.0097. Comparison between strains: WT (n = 27) versus atg-16.2 (n = 28), ****P < 0.0001, WT (n = 27) versus atg-4.1 (n = 24) ****P < 0.0001, over three independent experiments by two-way ANOVA with Tukey’s multiple-comparisons test. e,f, Lifespan analyses in sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals carrying atg-16.2(ok3224) (e) or atg-4.1(bp501) mutations (f) after whole-life atg-7, or lgg-1/ATG8 RNAi compared to control (CTRL). Two-sided log-rank test, NS P = 0.5, P = 0.6 (e), ****P < 0.0001 (f). See Supplementary Table 6 for details and repeats. g,h, Number of neuronal polyQ aggregates in day 5 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; rgef-1::Q40::yfp animals carrying atg-16.2(ok3224) (g) or atg-4.1(bp501) mutations (h) after RNAi treatments as in e. In the violin plots, the solid line indicates the median and dashed lines indicate quartiles. n = 45 animals each in three independent experiments (g) atg-16.2: NS P = 0.76, P = 0.46; (h) atg-4.1: ****P < 0.0001 by one-way ANOVA with Dunnett’s multiple-comparisons test. i, Mean percentage of ALMR with exophers of day 2 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; mec-4p::mCherry (WT) animals and atg-16.2(ok3224) animals after RNAi treatments as in e–h. Error bars are the s.d. of n = 7 experiments with n = 273, 255 and 257 animals (left) and n = 407, 411 and 357 animals (right). WT: ****P < 0.0001; atg-16.2: P = 0.62, P = 0.77 by two-sided Cochran–Mantel–Haenszel test.

Figure Legend Snippet: a, Mean percentage of ALMR neurons with exophers in day 2 WT, atg-16.2(ok3224) and atg-4.1(bp501) animals expressing mec-4p::mCherry. Error bars are the s.d. WT (n = 261 animals) versus atg-16.2 (n = 274 animals) (n = 7 experiments, **P = 0.007), WT (n = 273 animals) versus atg-4.1 (n = 243 animals) (n = 6 experiments, ****P < 0.0001) by two-sided Cochran–Mantel–Haenszel test. b, Mean GFP fluorescence intensity in head region in day 1 WT, atg-4.1(bp501) and atg-16.2(ok3224) animals expressing sqst-1p::sqst-1::gfp. Error bars are the s.d. WT (n = 30) versus atg-16.2 (n = 35), ****P < 0.0001; WT (n = 38) versus atg-4.1 (n = 58), ****P < 0.0001) by two-sided t-test over three independent experiments. Representative images from one experiment. Scale bar, 200 μm. c, Mean neuronal GFP::LGG-1/ATG8-positive and GFP::LGG-1(Gly116Ala)-positive punctae in day 1 WT, atg-4.1(bp501) and atg-16.2(ok3224) animals. Error bars are the s.d. WT-GFP::LGG-1 (n = 57) versus WT-GFP::LGG-1(Gly116Ala) (n = 61), ****P < 0.0001; atg-16.2-GFP::LGG-1 (n = 31) versus atg-16.2-GFP::LGG-1(Gly116Ala) (n = 32), ****P < 0.0001; atg-4.1-GFP::LGG-1 (n = 30) versus atg-4.1-GFP::LGG-1(Gly116Ala) (n = 28), NS P = 0.15. Comparison between strains: WT (n = 27) versus atg-16.2 (n = 31), ****P < 0.0001, WT (n = 30) versus atg-4.1 (n = 30) NS P = 0.75 > 0.05, ****P < 0.0001, over three independent experiments by two-way ANOVA with Tukey’s multiple-comparisons test. d, Mean neuronal GFP::LGG-1/ATG8-positive punctae in day 1 WT, atg-4.1(bp501) and atg-16.2(ok3224) animals after vehicle (DMSO) or BafA injections to block autophagy. Error bars are the s.d. WT-DMSO (n = 27) versus WT-BafA (n = 34), ****P < 0.0001; atg-16.2-DMSO (n = 28) versus atg-16.2-BafA (n = 34), *P = 0.043; atg-4.1-DMSO (n = 24) versus atg-4.1-BafA (n = 31), **P = 0.0097. Comparison between strains: WT (n = 27) versus atg-16.2 (n = 28), ****P < 0.0001, WT (n = 27) versus atg-4.1 (n = 24) ****P < 0.0001, over three independent experiments by two-way ANOVA with Tukey’s multiple-comparisons test. e,f, Lifespan analyses in sid-1; rgef-1p::sid-1 + rgef-1p::gfp animals carrying atg-16.2(ok3224) (e) or atg-4.1(bp501) mutations (f) after whole-life atg-7, or lgg-1/ATG8 RNAi compared to control (CTRL). Two-sided log-rank test, NS P = 0.5, P = 0.6 (e), ****P < 0.0001 (f). See Supplementary Table 6 for details and repeats. g,h, Number of neuronal polyQ aggregates in day 5 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; rgef-1::Q40::yfp animals carrying atg-16.2(ok3224) (g) or atg-4.1(bp501) mutations (h) after RNAi treatments as in e. In the violin plots, the solid line indicates the median and dashed lines indicate quartiles. n = 45 animals each in three independent experiments (g) atg-16.2: NS P = 0.76, P = 0.46; (h) atg-4.1: ****P < 0.0001 by one-way ANOVA with Dunnett’s multiple-comparisons test. i, Mean percentage of ALMR with exophers of day 2 sid-1; rgef-1p::sid-1 + rgef-1p::gfp; mec-4p::mCherry (WT) animals and atg-16.2(ok3224) animals after RNAi treatments as in e–h. Error bars are the s.d. of n = 7 experiments with n = 273, 255 and 257 animals (left) and n = 407, 411 and 357 animals (right). WT: ****P < 0.0001; atg-16.2: P = 0.62, P = 0.77 by two-sided Cochran–Mantel–Haenszel test.

Techniques Used: Expressing, Fluorescence, Comparison, Blocking Assay, Control

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Article Title: Autophagy protein ATG-16.2 and its WD40 domain mediate the beneficial effects of inhibiting early-acting autophagy genes in C. elegans neurons
Article Snippet: .. Autophagy flux assays were performed by injecting BafA (BioViotica) or vehicle (DMSO, Sigma) into animals expressing rgef-1p::gfp::lgg-1 or rgef-1p::gfp::lgg-1(Gly116Ala) 40 . ..

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