BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity Figure 7. bli-3 loss of function is hyper-resistant to Mn toxicity, insensitive to dopamine pre-treatment, and exhibits no dose- dependent Mn-associated ROS production. (A) bli-3(e767) is hyper-resistant (p,0.001) to Mn-induced lethality, with a LD50 = 83 mM. Pre-treatment with 10 mM dopamine (DA) does not change its hyper-resistance (p,0.001) and its LD50 = 84 mM is not significantly different from animals that were not pre-treated. (B) DA pre-treatment does not affect bli-3(e767) survival at 24 h. (C) Mn exposures from 1 mM up to 30 mM do not affect ROS levels significantly in bli-3(e767) worms (p.0.1). D) bli-3(e767) mutant does not show any significant difference in ROS levels upon Mn exposure up to 30 mM. doi:10.1371/journal.pgen.1001084.g007 show a significant difference in ROS production compared to dat- 1(ok157) worms at 3 mM (p,0.001) and compared to wild-type worms at 10 mM (p,0.01), but no difference compared to cat- 2(e1112) worms at any Mn concentration tested. These observations strongly support the notion that BLI-3 is required for the ROS-associated potentiating effect of DA in Mn toxicity. Given the structural analogy between DA and tyrosine, DA or DA-derived species formed upon reaction with Mn may serve as substrates for BLI-3, facilitating their conversion to highly oxidized species, and in turn, potentiating oxidative stress. Discussion This study establishes that Mn induces a specific and dose- dependent neurodegeneration of DAergic neurons in C. elegans, corroborating findings in rodents and non-human primates. This neurodegeneration was proven to require the DA re-uptake transporter, DAT-1, which is also the case in 6-OHDA-induced neurotoxicity. Combined, these observations suggest that upon Mn exposure, DAT-1 facilitates the intracellular transport of toxic species into DAergic neurons. Mn toxicity was associated with increased ROS generation and elevated F2IP levels as well as reduced longevity, all of which were aggravated by high endogenous DA levels and were attenuated by DA depletion. Consistent with a Mn-induced increase in oxidative stress, nuclear relocation of the anti-oxidant transcription factor, SKN-1, was noted in ASI neurons, and SKN-1 overexpression afforded protection against Mn-induced toxicity, while skn-1 deletion rendered worms more vulnerable to Mn toxicity. Increased extracellular, but not intracellular DA levels resulting from direct DA exposure or genetic manipulation potentiated Mn-induced lethality, oxidative-stress and reduction in lifespan. Finally, our studies establish that the dual-oxidase, BLI- 3, is involved in the DA-dependent Mn-induced toxicity and that bli-3 loss-of-function suppresses the DA-dependency of Mn toxicity and associated oxidative stress. Combined, these data suggest that oxidation of extracellular DA promoted by the BLI-3 dual-oxidase activity triggers DA-derived ROS generation, which, in turn, are taken up into the DAergic terminals by DAT-1 and lead to specific DAergic neurodegeneration (Figure 8). Relevance of our findings to the study of manganism and PD in mammalian models Mn-treated C. elegans recapitulate essential physiological aspects of parkinsonism, namely: the importance of NRAMP/DMT orthologues in the toxicity process [107,108,109]; the specificity PLoS Genetics | www.plosgenetics.org 10 August 2010 | Volume 6 | Issue 8 | e1001084