Direct in vivo imaging of ferrous iron dyshomeostasis in ageing Caenorhabditis elegans

Abstract

Iron is essential for eukaryotic biochemistry. Systematic trafficking and storage is required to maintain supply of iron while preventing it from catalysing unwanted reactions, particularly the generation of oxidising reactive species. Iron dyshomeostasis has been implicated in major age-associated diseases including cancers, neurodegeneration and heart disease. Here, we employ population-level X-ray fluorescence imaging and native-metalloproteomic analysis to determine that altered iron coordination and distribution is a pathological imperative of ageing in the nematode, Caenorhabditis elegans. Our approach provides a method to simultaneously study iron metabolism across different scales of biological organisation, from populations to cells. Here we report how and where iron homeostasis is lost during C. elegans ageing, and its relationship to the age-related elevation of damaging reactive oxygen species. We find that wild types utilise ferritin to sustain longevity, buffering against exogenous iron and showing rapid ageing if ferritin is ablated. After reproduction, escape of iron from safe-storage in ferritin raised cellular Fe²⁺ load in the ageing C. elegans, and increased generation of reactive species. These findings support the hypothesis that iron-mediated processes drive senescence. We propose that loss of iron homeostasis may be a fundamental and inescapable consequence of ageing that could represent a critical target for therapeutic strategies to improve health outcomes in ageing.

Fig. 1. Elevation and spatial redistribution of iron in ageing C. elegans. (a) Representative X-ray fluorescence micrographs of wild type adult C. elegans (4- and 12-day old). Inelastic scatter of incident photons (Compton scatter) provides anatomical visualisation (greyscale), and intestinal cells are highlighted by calcium (yellow). Iron (32-colour scale) was elevated throughout aged animals (scale bar = 100 μm). (b) Quantification of iron content in individual animals by XFM at intervals across lifespan. For all tested genotypes, 12- and 15-day old worms contained significantly more iron than 4-day old individuals (two-tailed unpaired Student's t-test; ** p < 0.01 and *** p < 0.001; mean ± SEM). Long-lived daf-2 mutants accumulated significantly less iron than age-matched wild type and daf-16(–);daf-2(–) (p < 0.001). (c) DAB-enhanced Perls' staining for non-heme iron (brown) in young (4-day, left) and aged (12-day, right) wild type C. elegans. Top row: Head sections. Middle row: Mid-body sections. Bottom row: Posterior sections. Insets show (i) intestinal cell nuclei free of iron in young C. elegans (filled triangles); (ii) discrete vesicular deposits of iron close to the intestinal lumen in young adults; (iii) iron in inclusions within the head of aged worms (filled arrowhead); (iv) dispersed and intranuclear iron (open triangles) within the intestine in aged individuals (scale bar = 100 μm).

Fig. 2. Elevated iron in C. elegans promotes oxidative stress in vivo. (a) Live imaging of exchangeable iron using calcein-AM fluorescence in (top) young (4-day) and (bottom) aged (12-day) C. elegans. Fluorescence is quenched by chemically accessible iron in the aged intestine. Bright field image (above) and fluorescence image (below) with an outline in white. (b) In vivo oxidative activity was detected by dichlorofluoroscein (DCF) fluorescence and increased in the intestine of old (bottom) compared to young (top) C. elegans. (c) Quantification of in vivo reactive species by DCF fluorescence. In the ageing worm, fluorescence significantly increased with age relative to 4-day old adults (mean ± SEM, n = 9, 7 and 5 individuals worms respectively; two-tailed unpaired Student's t-test, *** p < 0.001). (d) Ex vivo ROS generation is a product of iron accumulation. Long-lived daf-2 mutants lacked the increase in levels of reactive species with age (measured by DCF fluorescence; relative to 4-day old adults) compared to wild type and daf-16;daf-2 mutants (mean ± SEM, n = 4; two-tailed unpaired Student's t-test, *** p < 0.001).

Fig. 3. Iron storage capacity of ferritin is compromised during ageing. (a) Native soluble iron-binding species separated and detected by size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). In ageing C. elegans, greater levels of iron are associated with high molecular weight (Peak #1) and low molecular weight (Peak #3) species, whilst ferritin iron is decreased (Peak #2). (b) Integration of these three major chromatographic peaks across age (mean ± SEM, n = 3, one-way ANOVA with Dunnett's post hoc test, ** p < 0.01, *** p < 0.001). Linear regression analysis showed correlation between iron levels in each peak and age. (c) Loss of ftn-2 (null) ablated soluble ferritin-associated iron in young adults. In contrast, ftn-1 nulls did not differ from wild type, retaining the same iron-binding protein profile. (d) Representative X-ray fluorescence micrographs of wild type (i), ftn-1 null (ii) and ftn-2 null (iii) young adults confirmed loss of ftn-2 and resulted in a specific decrease in total iron with unaltered calcium levels. (e) Quantification of iron per adult revealed ftn-2 (ferritin) accounts for approximately 40% total iron in young C. elegans (mean ± SEM, n = 3, two-tailed unpaired Student's t-test, ** p < 0.01).

Fig. 4. Elevated iron alone does not decrease lifespan (a) exposure of young adult wild type C. elegans to high iron (as ferric ammonium citrate, FAC; 5 mg mL^–1) did not affect lifespan. In contrast, ftn-2 null mutants (16 day median lifespan) were significantly short-lived relative to wild type (18 day median lifespan; log-rank test, p < 0.001). The median lifespan of ftn-2 nulls was further reduced by exposure to FAC (15 day median lifespan; log-rank test, p < 0.001). (b) Ex vivo reactive species (as indicated by DCF fluorescence rate) generation was unaltered in lysates from 5-day old wild type exposed to high iron (5 mg mL^–1 FAC) for 48 h. Fluorescence rate from ftn-2 nulls raised on FAC was elevated relative to wild type (two-tailed unpaired Student's t-test, *** p < 0.001) and further elevated following iron exposure (two-tailed unpaired Student's t-test, p < 0.001). (c) Consistent with previously observed effects, wild type buffer high iron exposure (FAC) in ferritin, as shown by SEC-ICP-MS, whereas ftn-2 nulls were incapable.

Fig. 5. Ageing disturbs iron redox balance (a) integrated intensity of 71 X-ray fluorescence micrographs spanning the iron K-edge (7100 to 7170 eV; dashed grey lines) for the anterior portion of hydrated (i; n = 4) 5-day old wild type, (ii; n = 2) 5-day old ftn-2;ftn-1 nulls; and (iii; n = 2) 12-day old wild type adults. (b) Spatial distribution of ROIs defined by grouping pixels with similar spectra using k-means clustering. ROI-1 corresponds predominately to non-intestinal tissues, whilst ROI-2 through 5 are localized to the intestine. Scale bar = 100 μm. (c) Derivative iron K-edge spectra (∂XRF/∂Energy) for each ROI. Derivative spectra were modeled as the sum of two Gaussians between 7114 and 7134 eV (solid black lines) and the peak area associated with the 1s → 4s (light shaded curves) and 1s → 4p (dark shaded curves) transitions used to assess the Fe²⁺/Fe³⁺ balance of each ROI. Impaired ferritin function in aged wild type and ftn-2;ftn-1 null adults animals resulted in a proportional elevation of Fe²⁺.