no obvious increase in SKN-1::GFP intestinal expression was noted upon Mn exposure, possibly because SKN-1 was already over-expressed or because it is not activated by Mn. However, a notable change in the SKN-1::GFP localization pattern in the ASI neuron was associated with Mn exposure, where SKN- 1::GFP relocalized in discrete nuclear puncta, a result distinctly different from the diffuse pattern exhibited in non-Mn exposed worms (Figure 6B). This change was associated with a significantly increased average nuclear density of SKN-1::GFP (p,0.05, Figure 6C) but a non-significant increase in ASI expression, as revealed by the integral GFP intensity measure- ments (Figure 6D). These results support the notion that Mn exposure triggers an increase in ROS levels, thus activating the antioxidant response pathway, at least in ASI neurons. The data also indicate that the nuclear relocation of SKN-1, rather than its increased expression, is responsible for this protective effect against Mn toxicity. The dual-oxidase mutant bli-3(e767) is resistant to DA-dependent Mn toxicity Given that Mn induces DA-dependent oxidative stress, we next sought to identify genetic factors involved in this process. In BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity addition to the mitochondrial electron-transport chain (ETC), plasma membrane NADPH oxidases are major contributors to ROS production in rotenone, paraquat and MPP+-induced toxicity [86,100,101,102,103]. The bli-3 gene encodes a C. elegans dual-oxidase orthologue to vertebrates DUOX1 and DUOX2, which is involved in the di-tyrosine bond formation in the worm cuticle [104] and pathogen-induced ROS production [105,106]. Di-tyrosine bonds maintain cuticle integrity, and bli-3(e767) mutants display a blistered cuticle and a mild dumpy phenotype. This phenotype renders worms more sensitive to toxicants due to increased absorbance of the toxicants through the damaged cuticle [104]. Interestingly, bli-3(e767) worms displayed hyper-resistance to acute Mn treatments when compared to wild-type worms (p,0.001), with a LD50 = 83 mM (Figure 7A), suggesting that BLI- 3 is involved in mediating Mn-toxicity, most likely by potentiating ROS production and oxidative stress. Notably, sub-lethal (Figure 7B) DA pre-treatment did not affect bli-3(e767) worm sensitivity to Mn: LD50 = 84mM (Figure 7A). Moreover, similar to cat-2(e1112) mutants (Figure 5B), bli-3(e767) mutants did not show any dose-dependent increase in ROS production from 1 mM to 30 mM Mn exposures (Figure 7C). ANOVA (comparing data from Figure 5B and Figure 7C) reveals that bli-3(e767) mutants Figure 6. skn-1 mutants are hypersensitive while SKN-1::GFP overexpressing worms are hyper-resistant to Mn toxicity and respond to Mn exposure by relocalizing SKN-1::GFP to nuclear puncta in ASI neurons. (A) SKN-1::GFP expressing worms are hyper-resistant (p,0.001) to Mn-induced lethality, with a LD50 = 114 mM, while mixed skn-1(ok2315) and skn-1(ok2315)/+ are hypersensitive (p,0.001) to Mn exposure, with a LD50 = 34 mM. (B) SKN-1::GFP nuclear localization in ASI neurons reorganizes in puncta 1 hour after MnCl2 exposure. (C) SKN-1::GFP mean brightness significantly increases (p,0.05) in ASI nuclei upon Mn exposure while integral brightness does not (D). doi:10.1371/journal.pgen.1001084.g006 PLoS Genetics | www.plosgenetics.org 9 August 2010 | Volume 6 | Issue 8 | e1001084