BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity Figure 5. Dopamine-dependent Mn toxicity is associated with decreased lifespan and increased oxidative stress. (A) cat-2(e1112) and dat-1(ok1057) mutants both show a reduced lifespan (p,0.001) upon Mn acute treatment. dat-1(ok1057) exhibit a further reduced lifespan, whereas cat-2(e1112) show a significant improvement upon 3 mM MnCl2 acute treatment and a further increased lifespan (p,0.001) upon 100 mM MnCl2 acute treatment. Plotted values represent averages of three independent experiments and the curves represent the best sigmoidal fit. (B) Wild-type worms exhibit significantly higher reactive oxygen species (ROS) levels upon 10 mM Mn treatment (p,0.05), while dat-1(ok157) mutant show a significant increase after only 1 mM Mn exposure (p,0.01), and cat-2(e1112);dat-1(ok157) display normal ROS levels up to 30 mM Mn exposure. (C) Wild-type worms display significantly increased isoprostane-2 (F2IP) levels upon 10 mM Mn treatment (p,0.001), while dat-1(ok157) mutant show a significant increase after 3 mM Mn exposure (p,0.001), and cat-2(e1112);dat-1(ok157) upon 20 mM (p,0.05) and 30 mM (p,0.01). doi:10.1371/journal.pgen.1001084.g005 strongly suggest that the DA-dependent Mn-induced toxicity involves oxidative stress. The anti-oxidant transcription factor, SKN-1, protects against Mn toxicity and relocalizes in amphide sensilla ASI neuron nuclei upon Mn exposure An alternative functional way to establish oxidative stress, especially in its earliest stages, is to demonstrate a physiological response to it. Sodium arsenite exposure in C. elegans has previously been shown to induce a strong intestinal expression of the anti-oxidant response gene, skn-1, the orthologue of the vertebrate gene, Nrf2 [93]. SKN-1 initiates the development of the digestive system and feeding during the earliest embryonic stages, and post-embryonically, is required for a normal lifespan and adequate resistance to stress [94,95,96]. Consistent with these observations, skn-1 deletions or loss-of-function mutations suppress oxidative stress resistance [97]. Aging in C. elegans is delayed when SKN-1 is transgenically expressed, and a mutant skn-1 form that constitutively localizes to nuclei increases the worm’s lifespan [94,97,98,99]. To determine if SKN-1 can afford protection against Mn toxicity and to confirm that oxidative stress plays an important role in Mn-induced toxicity in C. elegans, SKN-1::GFP over-expressing worms were exposed to Mn. These worms exhibited a strong hyper-resistant phenotype to Mn exposure with a LD50 = 114 mM (Figure 6A). This effect was significantly greater in comparison to wild-type (p,0.001) or GFP expression alone (p,0.01) under various promoters (cf. dat- 1::GFP, Figure 2; data not shown) despite the aforementioned protective effect of GFP expression, suggesting that SKN-1 directly protects against Mn toxicity. Moreover, a mixed population of two-third heterozygous and one-third homozygous deletion-mutant skn-1(ok2315) expressing a truncated SKN-1 protein, exhibited increased sensitivity to Mn exposure with a LD50 = 34 mM (Figure 6A), confirming that SKN-1 is required for mediating optimal resistance to Mn exposure. Unexpectedly, PLoS Genetics | www.plosgenetics.org 8 August 2010 | Volume 6 | Issue 8 | e1001084