BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity Figure 2. dat-1 loss of function induces an overall hypersensitivity to Mn toxicity, but confers neuroprotection to the dopaminergic system. (A) Most surviving dat-1::GFP expressing dat-1(ok157) mutants fail to exhibit dopaminergic neurodegeneration, while surviving dat-1::GFP expressing wild-type worms always display dopaminergic processes loss and cell body shrinkage upon 150 mM Mn acute exposure. (B) dat-1::GFP expressing dat-1(ok157) mutants are hypersensitive (p,0.001) to Mn exposure, with a LD50 = 17 mM. C) dat-1(ok157) is hypersensitive (p,0.001) to Mn-induced lethality, with a LD50 = 9 mM. doi:10.1371/journal.pgen.1001084.g002 paralysis (SWIP) phenotype, likely due to the hyper-activation of DA-responsive motorneurons exposed to excessive synaptic DA concentrations [71]. C. elegans dat-1 is also required for 6-OHDA DAergic neuron toxicity as noted in dat-1::GFP transgenic worms [56]. Given the specific neurodegeneration of dat-1::GFP-express- ing neurons upon 6-OHDA or Mn exposure, we hypothesized that dat-1 is required for Mn-induced DAergic degeneration. Accord- ingly, dat-1(ok157) worms expressing dat-1::GFP were exposed to graded Mn doses in parallel with wild-type worms expressing the same dat-1::GFP array, and both strains were scored for DAergic neuron defects. Mn treatment was associated with marked DAergic neurodegeneration in wild-type worms, while dat-1 mutant DAergic neurons were not significantly affected even at the highest doses of Mn (Figure 2A) exposure for which fewer than 5% of the worms survived (Figure 2B). Accordingly, dat-1 mutant worms exposed to Mn most likely died from osmoregulation defects before displaying any neurodegeneration in DAergic neurons. These results established that DAergic neurodegenera- tion in Mn-exposed worms requires a functional DAT-1 transporter. PLoS Genetics | www.plosgenetics.org 4 August 2010 | Volume 6 | Issue 8 | e1001084