Zinc Levels Modulate Lifespan through Multiple Longevity Pathways in <i>Caenorhabditis elegans</i>

<div><p>Zinc is an essential trace metal that has integral roles in numerous biological processes, including enzymatic function, protein structure, and cell signaling pathways. Both excess and deficiency of zinc can lead to detrimental effects on development and metabolism, resulting in abnormalities and disease. We altered the zinc balance within <i>Caenorhabditis elegans</i> to examine how changes in zinc burden affect longevity and healthspan in an invertebrate animal model. We found that increasing zinc levels <i>in vivo</i> with excess dietary zinc supplementation decreased the mean and maximum lifespan, whereas reducing zinc levels <i>in vivo</i> with a zinc-selective chelator increased the mean and maximum lifespan in <i>C</i>. <i>elegans</i>. We determined that the lifespan shortening effects of excess zinc required expression of DAF-16, HSF-1 and SKN-1 proteins, whereas the lifespan lengthening effects of the reduced zinc may be partially dependent upon this set of proteins. Furthermore, reducing zinc levels led to greater nuclear localization of DAF-16 and enhanced dauer formation compared to controls, suggesting that the lifespan effects of zinc are mediated in part by the insulin/IGF-1 pathway. Additionally, zinc status correlated with several markers of healthspan in worms, including proteostasis, locomotion and thermotolerance, with reduced zinc levels always associated with improvements in function. Taken together, these data support a role for zinc in regulating both development and lifespan in <i>C</i>. <i>elegans</i>, and that suggest that regulation of zinc homeostasis in the worm may be an example of antagonistic pleiotropy.</p></div

TPEN effects on <i>C</i>. <i>elegans</i> metal content and lifespan are zinc-specific.

<p><b>(A) TPEN effect on labile zinc levels is specific to the chelation of zinc</b>. Wildtype worms were exposed to 200μM TPEN in the presence or absence of 500μM ZnSO₄ or 500μM MgSO₄ at L3 stage and collected at one-day old adult animals for analysis of relative labile zinc content. The representative micrograph shows that worms supplemented with zinc have elevated labile zinc content, while TPEN treatment decreased labile zinc. Equal molar levels of magnesium had no effect on basal or TPEN-induced fluorescence. Scale bar = 0.2mm. <b>(B) Kaplan-Meier survival curve of worm populations exposed to zinc and/or TPEN</b>. Wildtype worms were exposed to 200μM TPEN in the presence or absence of 500μM ZnSO₄ at the L3 stage and monitored for effects on lifespan. Mean and maximum population lifespan were reduced with zinc supplementation and increased with TPEN treatment (p < 0.0001, log rank test). Additional 500μM ZnSO₄ attenuated the effect of TPEN on mean and maximum lifespan. <b>(C) Kaplan-Meier survival curve of worm populations exposed to magnesium and/or TPEN</b>. Wildtype worms were exposed to 200μM TPEN in the presence or absence of 500μM MgSO₄ at the L3 stage and monitored for effects on lifespan. Mean and maximum population lifespan were increased with TPEN treatment (p < 0.0001, log rank test). Addition of MgSO₄ had no effect on mean and maximum lifespan.</p

Zinc availability alters dauer formation, DAF-16 localization, and lifespan of insulin signaling pathway mutants in <i>C</i>. <i>elegans</i>.

<p><b>(A) Kaplan-Meier survival curve of worm populations exposed to zinc or TPEN</b>. Worms with null alleles for <i>daf-2</i> were exposed to 500μM ZnSO₄ or 200μM TPEN at L4 stage animals. Mean and maximum population lifespan were reduced with zinc and increased with TPEN treatment (p < 0.0001, log rank test). <b>(B) Changes in dauer formation in worm populations exposed to zinc or TPEN as larvae</b>. Worms with null alleles for <i>daf-2</i> were exposed to 500μM ZnSO₄ or 200μM TPEN at L3 stage and collected at one-day old adult animals and analyzed for dauer formation. Worms supplemented with zinc had a reduced number of worms in the dauer state, whereas worms treated with TPEN had an elevated number of worms in the dauer state (**, p<0.001, ***, p<0.0001, t-test). Data indicates the mean ± SD of 3 independent experimental replicates. <b>(C) Imaging DAF-16 localization in worm populations exposed to zinc or TPEN at 22°C</b>. DAF-16::GFP transgenic worms were exposed to 500μM ZnSO₄ or 200μM TPEN at L3 stage and measured for DAF-16 localization of one-day old adult animals. Representative micrographs showed that the subcellular localization of DAF-16::GFP was more evident in the nucleus when worms were exposed to TPEN, compared to control and zinc-supplemented worms. Scale bar = 200μm. <b>(D) Quantifying DAF-16 localization in worm populations exposed to zinc or TPEN at 22°C</b>. GFP fluorescence from DAF-16::GFP transgenice worms was imaged by fluorescence microscopy and scored for elevated nuclear DAF-16 localization. Data is represented as mean ± S.D using results of 3 experimental replicates (***, p<0.0001, log rank test). <b>(E) Imaging DAF-16 localization in worm populations exposed to zinc or TPEN at 34°C</b>. DAF-16::GFP transgenic worms were exposed to 500μM ZnSO₄ or 200μM TPEN at L3 stage and measured for DAF-16 localization of one-day old adult animals, after exposure to heat shock at 34°C for 5 min. Representative micrographs showed that the subcellular localization of DAF-16::GFP was reduced in the zinc treated group and elevated in in the TPEN group, compared to control worms. Scale bar = 200μm. <b>(F) Quantifying DAF-16 localization in worm populations exposed to zinc or TPEN at 34°C</b>. GFP fluorescence from DAF-16::GFP transgenic worms was imaged by fluorescence microscopy and scored for elevated nuclear DAF-16 localization. Data is represented as mean ± SD using results of 3 experimental replicates (***, p<0.0001, log rank test)</p

Identification of genes that modulate the effects of zinc and TPEN on lifespan in <i>C</i>. <i>elegans</i>.

<p>(A) Wildtype worms, or worms with null alleles for <b>(B)</b> <i>hsf-1(sy441)</i>, <b>(C)</b> <i>daf-16(mu86)</i>, <b>(D)</b> <i>aak-2(ok524)</i>, <b>(E)</b> <i>rsks-1(ok1255)</i>, <b>(F)</b> <i>nhr-49(ok2165)</i>, <b>(G)</b> <i>skn-1(eu31)</i>, and <b>(H)</b> <i>clk-1(e2519)</i> were exposed to 500μM ZnSO₄ or 200μM TPEN at the L3 stage. Mean and maximum population lifespan were reduced with zinc supplementation and increased with TPEN treatment for many mutant strains, similar to wildtype worms (p < 0.0001, log rank test). However, ZnSO₄ and TPEN-mediated changes in lifespan were attenuated in worms with mutations of <i>daf-16(mu86)</i>, <i>hsf-1(sy441)</i>, <i>skn-1(eu31)</i>.</p

Zinc availability modulates healthspan in <i>C</i>. <i>elegans</i>.

<p><b>(A) Levels of age-related protein aggregation in worm populations exposed to zinc or TPEN</b>. L4 stage TJ1060 [spe-9(hc88)I; fer-15(b26)II] worms were exposed to 500μM ZnSO₄ or 200μM TPEN at L3 stage for 6 days and then analyzed for SDS-insoluble protein. The representative experiment shows worms exposed to TPEN had reduced SDS-insoluble protein in comparison with control animals. (M, marker; LC, loading control of control worms; TC, loading control of TPEN treated worms; IC, SDS-insoluble protein of control animals; IT; SDS-insoluble protein of TPEN treated worms.) <b>(B) Percent paralysis of worms with knockdown for specific genes supplemented with or without TPEN</b>. HE250 worms with knockdown for <i>hsf</i>-1(RNAi), <i>skn</i>-1(RNAi), or <i>daf</i>-16(RNAi) and with or without exposure to 200μM TPEN were scored for paralysis after 48 hours. Control worms treated with 200μM TPEN showed significantly less paralysis in comparison to control animals (***, p<0.0001, log rank test). However, TPEN was less effective when used in populations with knockdown in <i>hsf</i>-1, <i>skn</i>-1, or <i>daf</i>-16 genes (NS, not significant).</p

Zinc availability regulates the lifespan of <i>C</i>. <i>elegans</i>.

<p><b>(A) Kaplan-Meier survival curve of worm populations exposed to zinc or TPEN as larvae</b>. Wildtype worms were exposed to 500μM ZnSO₄ or 200μM TPEN at the L3 stage. Mean and maximum population lifespan were reduced with zinc and increased with TPEN treatment (p < 0.0001, log rank test) <b>(B) Kaplan-Meier survival curve of worm populations exposed to zinc or TPEN as adults</b>. Wildtype worms were exposed to 500μM ZnSO₄ or 200μM TPEN at day 5 of adulthood. Thl.ere was no significant difference in mean and maximum population lifespan due to zinc or TPEN treatment. <b>(C) Changes in total zinc content in worms exposed to zinc or TPEN as larvae</b>. Wildtype worms were exposed to 500μM ZnSO₄ or 200μM TPEN at the L3 stage and collected at one-day old adult animals for analysis of total zinc content. Worms supplemented with zinc had a ~2-fold increase in total zinc content (***, p<0.0001, t-test), while worms treated with TPEN demonstrated a ~2-fold decrease in total zinc content (**, p<0.001, t-test). Data shown are the mean ± SD of 3 experimental replicates.</p

The effects of zinc availability on lifespan is dependent on HSF-1, SKN-1, and DAF-16 transcription factors in <i>C</i>. <i>elegans</i>.

<p><b>(A) Kaplan-Meier survival curve of worms with knockdown of <i>hsf-1</i> exposed to TPEN as larvae</b>. <i>hsf-1</i> knockdown worms with or without knockdown of <i>skn-1</i> were exposed to 200μM TPEN at the L3 stage. The effect of TPEN on lifespan in this mutant was reduced when <i>skn-1</i> was also knockdown (p < 0.0001, log rank test). <b>(B) Kaplan-Meier survival curve of worms with knockdown of <i>hsf-1</i> exposed to TPEN as larvae</b>. <i>skn-1</i> knockdown worms with or without knockdown of <i>hsf-1</i> were exposed to 200μM TPEN at the L3 stage. The effect of TPEN on lifespan in this mutant was reduced when <i>hsf-1</i> was also knockdown (p < 0.0001, log rank test). <b>(C) Kaplan-Meier survival curve of worms with knockdown of <i>daf-16</i> exposed to TPEN as larvae</b>. <i>daf-16</i> knockdown worms with or without knockdown of <i>hsf-1</i> were exposed to 200μM TPEN at the L3 stage. The effect of TPEN on lifespan in this mutant was reduced when <i>hsf-1</i> was also knockdown (p < 0.0001, log rank test). <b>(D) Kaplan-Meier survival curve of worms with knockdown of <i>daf-16</i> exposed to TPEN as larvae</b>. <i>daf-16</i> knockdown worms with or without knockdown of <i>skn-1</i> were exposed to 200μM TPEN at the L3 stage. The effect of TPEN on lifespan in this mutant was reduced when <i>skn-1</i> was also knockdown (p < 0.0001, log rank test).</p