Treatment with DEM represents an acute stress that reduces glutathione levels and causes death, with older animals being more susceptible.
Note, the age of adults is determined by the number of days following the last larval molt and therefore reflects the number of days of adulthood. (A) Survival curves of adults following either 24- or 48 hr exposure to increasing doses of DEM. Treatment begun on Day 4 of adulthood. Shown are proportions ± 95% confidence intervals (Newcombe, 1998), with a sigmoidal curve fitted. (B) Total glutathione (GSH) decrease following 6 hr of DEM exposure. Day 4 adults used, with results normalized to the GSH levels in worms not exposed to DEM. Plotted are six independent replicates, with each estimated derived from 50 adults per measure). Linear regression R2 = 0.98, p=0.01 (C) Total GSH levels decrease with increased adult age in C. elegans. Each point is derived from six independent replicates of 50 adults, with black lines marking the mean value. Results are normalized to the GSH levels in Day one worms (ANOVA: F (3, 20)=32.96, p<0.0001; see Supplementary file 1 for pairwise comparisons). (D) Aged C. elegans adults become progressively more sensitive to GSH depletion by DEM. Shown are proportions ± 95% confidence intervals, with a sigmoidal curve fitted. (E) Both Lip and SIH treatment protect against lethality from DEM-derived glutathione depletion. Day 4 adults, with values representing pooled data from four independent experiments ± 95% confidence intervals, each with a fitted sigmoidal curve. Ctl denotes vehicle control (0.5% v/v DMSO). Pairwise comparisons at 10 and 20 mM DEM were performed using Fisher’s exact test; *** denotes p<0.001. (F) Total glutathione levels are preserved following SIH pretreatment from L4, but not by Lip-1. Day 4 adults were exposed to DEM (10 mM) for 6 hr, and total glutathione (GSH) assayed. Ctl denotes vehicle control (0.5% v/v DMSO). Each point is derived from 4 to 9 independent replicates of 50 adults, with black lines marking the mean value ± SD. (ANOVA: F (5, 30)=50.97, p<0.0001; see Supplementary file 1 for pairwise comparisons). *** denotes p<0.001.
Data for DEM and GSH comparisons.
Measures of lipid peroxidation and cell death show an increase with age and reduction by both Lip-1 and SIH treatment.
In all panels, vehicle control (0.5% v/v DMSO, Ctl)-treated worms are shown in orange, Lip-1-treated (200 µM Lip-1) are green and SIH-treated (250 µM SIH) are blue. (A) Representative propidium iodide fluorescence (red) overlay of bright field micrograph depicting dead intestinal cells (marked by nuclear signal, yellow triangles) within a live Day 4 adult treated with DEM. Smaller fluorescent puncta were also observed, consistent with neuronal cell nuclei (white unfilled triangles). Untreated Day 4 adult control animals (Ctl) showed no cell death. (B) Proportion of live animals at Day 4 (±95% confidence intervals) of adulthood showing dead cell fluorescence (propidium iodide)±exposure to 10 mM DEM for 24 hr. Cohorts of animals included: co-treatment with a vehicle control (-DEM, Ctl, n = 102; +DEM Ctl n = 117), Lip-1 (-DEM, Lip-1, n = 84; +DEM Ctl n = 106) or SIH (-DEM, Ctl, n = 89; +DEM Ctl n = 129). Lip-1 and SIH both markedly reduced the proportion of animals with dead cells after DEM treatment (z-test: Ctl vs Lip-1 Z = 6.37 ***p<0.001; Ctl vs SIH Z = 7.24, ***p<0.001). (C) Proportion of live animals at 4, 6 and 8 days of adulthood (±95% confidence intervals) showing propidium iodide nuclear fluorescence. Day 6 Ctl adults (n = 160) had a significantly higher proportion of animals with dead cells than Day 4 Ctl (n = 263; z-test: Z = 4.70 ***p<0.001). Similarly, Day 8 Ctl adults (n = 119) had significantly higher prevalence of animals with propidium iodide nuclear fluorescence than Day 4 Ctl (z-test: Z = 6.65 ***p<0.001). No significant difference was observed between Day 6 and Day 8 control populations. Within the Day 6 adult cohorts treatment with Lip-1 (n = 77) or SIH (n = 81) markedly reduced the prevalence of animals with propidium iodide nuclear fluorescence compared to vehicle control (z-test: Ctl vs Lip-1 Z = 2.57 # p<0.001; Ctl vs SIH Z = 2.64, # p<0.001). Within the Day 8 adult cohorts treatment with Lip-1 (n = 234) or SIH (n = 308) markedly reduced the prevalence of animals with propidium iodide nuclear fluorescence compared to vehicle control (Ctl, n = 119; z-test: Ctl vs Lip-1 Z = 5.67 # p<0.001; Ctl vs SIH Z = 6.28, # p<0.001). (D) Levels of the lipid peroxidation end product malondialdehyde (MDA) increases in C. elegans following acute glutathione depletion by 20 mM DEM exposure for 6 hr. MDA levels are shown as values normalized against the mean of untreated Day 4 Adults (Ctl) for independent samples. (Ctl vs +DEM, unpaired 2-tailed t-test ***p<0.001) (E) Malondialdehyde (MDA) increases in aged C. elegans (Day 1 vs Day 8 adults, ANOVA *p<0.05). Treatment with either Lip-1 (Day 8 vs Day 8 +Lip-1 adults, ANOVA ***p<0.001) or SIH (Day 8 vs Day 8 +SIH adults, ANOVA *p<0.05) reduces levels of MDA. Data represent independent samples with values normalized against the mean of untreated Day 1 adults. (F) Representative immunoblot against 4-HNE protein adducts comparing Day 1 and Day 8 control adults and aged adults treated with Lip-1 and SIH with corresponding tubulin blot below (representative of triplicate experiments). The relative intensity of the bands show an age-related increase that is ameliorated by Lip-1 and SIH.
Data for cell death and lipid peroxidation.
Representative images are shown in A for DEM-treated animals and B for aging animals. (A) Representative epifluorescence micrographs of dead cell fluorescence (propidium iodide) in animals ± exposure…
Images of control (Ctl), 200 µM Lip-1 and 250 µM SIH-treated animals are shown in A to C. DEM-treated (±10 mM DEM) animals are shown in D and E. Lip-1 and SIH do not cause nuclear translocation of …
In all panels, vehicle control (0.5% v/v DMSO, Ctl) treated worms are shown in orange, Lip-1 treated (200 µM Lip-1) are green and SIH treated (250 µM SIH) are blue. (A) Representative X-ray …
Data for XFM and mass spectrometry comparisons.
𝜑XANES evaluation of the Fe2+ fraction in vivo within intact animals. (A) Schematic highlighting the anatomy of an adult hermaphrodite C. elegans with the intestine shaded in grey. Dashed box is …
Data for XANES comparisons.
Similarly, pooled spectra and first derivatives from 4-day-old adult wild types treated with (n = 4) and without (n = 4) DEM is shown in Fig C-D Changes in the first derivative of the Fe K-edge …
Treatment with both Lip-1 and SIH extend lifespan. (A) Representative Kaplan-Meier survival curve from C. elegans treated with vehicle control (Ctl, median survival 7 days, death events n = 88); …
Data for survival comparisons.
(A) Median survival (Log-rank (Mantel-Cox) test: Control = 6 days; 25 μM Lip-1 = 6 days (p=0.07 ns); 100 μM Lip-1 = 6 days (p=0.006); 200 μM Lip-1 = 12 days (p<0.0001); 250 μM SIH = 13 days (p<0.0001…
To determine whether the increased lifespan seen with SIH treatment could be explained solely by an antibiotic effect of iron reduction, nematodes were treated with ampicillin, with and without SIH …
In all panels, vehicle control (0.5% v/v DMSO, Ctl)-treated worms are shown in orange, Lip-1-treated (200 µM Lip-1) are green and SIH-treated (250 µM SIH) are blue.
Significant differences between treatments are highlighted where * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001. (A) Micrograph of an adult C. elegans on the first day of treatment (Day 0) and animals treated with Ctl, Lip-1 or SIH for eight days at 25°C (Day 8). Scale bar = 50 µm. (B) Estimates of adult body length (in µm), showing SIH- treated animals (blue) have longer average body length compared to age matched control (orange)or Lip-1 (green) treated populations (Kruskal-Wallis ANOVA: H(10) = 432.6, p<0.0001; see Supplementary file 6 for sample summary and pair-wise comparisons). Start (grey) represents the beginning population of L4/young adults grown from egg at 25°C for 48 hr prior to transfer to treatment plates. Each point represents an individual worm, with mean and error bars representing standard deviation (SD) (C) Estimated adult body volume (in pL), showing increased body volume with adult age for all groups (Kruskal-Wallis ANOVA: H(10) = 489, p<0.0001; see Supplementary file 6 for sample summary and pair-wise comparisons), with SIH treated animals having even greater body volume. Each point represents an individual worm, with mean ± SD. (D) Early fertility (first 24 hr) and total reproductive output are unaltered when vehicle control (Ctl)-treated cohorts are compared to Lip-1 or SIH-treated animals at 25°C. Each data point represents an estimate from a single C. elegans adult, with mean ± SD. Sample size: early fertility Ctl n = 25, Lip-1 n = 26, SIH n = 26; total Ctl n = 21, Lip-1 n = 17, SIH n = 19 (ANOVA: Early fertility F(2,74)=0.996, p=0.37; Total fertility F(2,57) = 4.89, p=0.011). (E) Estimates of maximum velocity achieved by aged and treated cohorts of C. elegans. Treatment with either Lip-1 or SIH attenuates the age-related decline in maximum velocity (Kruskal-Wallis ANOVA: H(7) = 298.5, p<0.0001; see Supplementary file 6 for sample summary and pair-wise comparisons). Each data point represents an estimate from a single C. elegans adult, with median ±interquartile range. Equivalent analyses of mean velocity and total distance travelled (and how these data correlate) are shown in Figure 6—figure supplement 1 and Supplementary file 6).
Data for fertility, size and movement.
Movement parameters measured included maximum velocity (shown in Figure 6E), mean velocity and total distance travelled. The latter two datasets are represented in Figure 6-figure supplement A and …
During normal aging iron unnecessarily accumulates. The safe storage of surplus iron in ferritin begins to fail in late life, causing a corresponding elevation of reactive, ‘labile’ iron. In …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Caenorhabditis elegans, hermaphrodite) | Wild type | Caenorhabditis Genetics Center (CGC) | N2 | |
Strain, strain background (Caenorhabditis elegans, hermaphrodite) | spe-9(hc88); rrf-3(b26) | Caenorhabditis Genetics Center (CGC) | TJ1060 | temperature sensitive-sterile strain |
Strain, strain background (Caenorhabditis elegans, hermaphrodite) | zIs356 [Pdaf-16::daf-16a/b::gfp + rol-6(su1006)] | Caenorhabditis Genetics Center (CGC) | TJ356 | DAF-16 reporter strain |
Antibody | 4-HNE protein adduct antibody (goat polyclonal) | Millipore | Cat.#: AB5605 | (1:2000 dilution) |
Antibody | anti-Tubulin antibody (mouse monoclonal) | Sigma-Aldrich | Cat.#: T6074 | (1:10,000 dilution) |
Commercial assay or kit | Thiobarbituric acid reactive substances (TBARS) assay kit | Caymen Chemical | Cat.#: 10009055 | |
Commercial assay or kit | BCA Protein assay kit | Pierce | Cat.#: 23225 | |
Chemical compound, drug | Diethyl maleate | Sigma-Aldrich | DEM Cat.#: D97703-100G | |
Chemical compound, drug | Liproxstatin | Marcus Conrad (Helmholtz Zentrum München; initially) and subsequently ApexBio Tech LLC | Lip-1 | N-[(3-chlorophenyl) methyl]-spiro[piperidine-4,2'(1'H)-quinoxalin]−3'-amine) |
Chemical compound, drug | Salicylaldehyde isonicotinoyl hydrazone | Des Richardson (University of Sydney) | SIH | |
Software, algorithm | GeoPIXE 7.3, Dynamic Analysis | CSIRO | Ryan, 2000. Quantitative trace element imaging using PIXE and the nuclear microprobe. Int J Imag Syst Tech 11, 219–230. | |
Software, algorithm | ImageJ WormSizer plugin | Moore, B.T., Jordan, J.M., and Baugh, L.R. (2013). WormSizer: high-throughput analysis of nematode size and shape. PLoS One 8, e57142. | ||
Software, algorithm | Prism v7.0a, GraphPad Software | www.graphpad.com | ||
Software, algorithm | R package flexsurv | rdocumentation.org/packages/flexsurv | ||
Software, algorithm | Image J, wrMTrck plugin | Nussbaum-Krammer, C.I., Neto, M.F., Brielmann, R.M., Pedersen, J.S., and Morimoto, R.I. (2015). J Vis Exp, 52321 | ||
Other | propidium iodide | Life Technologies | P3566 | 0.25 mg/mL solution |
Statistical analysis for Figure 1.
Statistical analysis for Figure 3.
Statistical analysis for Figure 4 and Figure 4—figure supplement 2.
Statistical analysis for Figure 5.
Details for temporal scaling analysis associated with Figure 5.
Statistical analysis for Figure 6.
The figures showing the ROIs associated with each iron map (ROI; white = overlapped/fractional, green = whole/minimally overlapped, red = excluded from analysis).
Shown are the masks used to identify and analyse the iron elemental maps of TJ1060 populations at different adult ages ± Lip-1 or SIH at 25°C. (A) Masks for 1 day old adults (starting population) (B) Masks for 4 day old Control adults (C) Masks for 4 day old SIH-treated adults (D) Masks for 4 day old Lip-1 treated adults E: Masks for 8 day old Control adults (F) Masks for 8 day old 250 µM SIH-treated adults (G) Masks for 8 day old 200 µM Lip-1-treated adults.