dat-1(ok157) mutant worms are hypersensitive to Mn-induced toxicity Additional studies showed that the protective effect of dat-1 loss- of-function on DAergic neurodegeneration was not due to an indirect effect, such as an adaptive mechanism resulting in reduced Mn-uptake. First, the lethal dose 50 at 24 h (LD50, dose of Mn exposure at which 50% of the animals die from the treatment) for both transgenic strains, dat-1(ok157); dat-1::GFP and dat-1(+); dat- 1::GFP, was determined. Notably, the dat-1(ok157); dat-1::GFP strain showed significant hypersensitivity (p,0.001) to Mn- induced toxicity with a LD50 = 20 mM, whereas the dat-1(+); dat-1::GFP strain exhibited a LD50 = 74 mM (Figure 2B). Next, the non-transgenic dat-1(ok157) and the N2 wild-type strains were treated with the same range of Mn doses (0.001 mM to 1 M). Both non-transgenic strains were slightly more sensitive than the corresponding dat-1::GFP-expressing strains (Figure 2C). This ‘‘protective effect’’ of the transgene expression was not specific to the dat-1::GFP construct and was systematically observed with any type of transcriptional GFP-expressing construct (data not shown). Transgenic worms may experience higher basal levels of stress associated with increased stress-response protein levels, enabling them to better cope with environmental stress. Never- theless, dat-1(ok157) mutants exhibited hypersensitivity to Mn- induced lethality (p,0.001), which was characterized by a LD50 = 9 mM as compared to a LD50 = 47 mM for wild-type worms (Figure 2C). This observation rules out the possibility that dat-1(ok157) takes up less Mn. Moreover, it shows that upon Mn exposure, DAT-1 loss-of-function is detrimental to worm survival, though protective to DAergic neurons. Sub-lethal exogenous DA pre-treatment potentiates Mn toxicity The importance of a functional DAT-1 in conferring selective neurodegeneration in C. elegans DAergic neurons upon acute Mn exposure reflected upon earlier work with the PD-mimicking drug and DA analogue, 6-OHDA. Indeed, 6-OHDA induced a dose- dependent DAergic neurodegeneration in C. elegans, which was prevented by DAT-1 mutations [56,72]. The fact that both Mn and 6-OHDA induce neurotoxicity through DAT-1 raises a question regarding the relationship between Mn and DA BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity analogues upstream of DAT-1 in the DAergic neurodegeneration pathway. It has been hypothesized that Mn, in its divalent or trivalent cationic form, like iron, reacts with biogenic amines, such as DA, to generate ROS [73,74,75]. It is also known that DA and its derivatives, such as L-DOPA and 6-OHDA, can be toxic to the mammalian DAergic system and can lead to intracellular oxidative stress alone or in combination with metals and ensuing DAergic neurodegeneration [46,76,77]. Accordingly, we hypothesized that DA and Mn have a synergistic toxic effect in the worm. To test this hypothesis, worms were acutely treated with 10 mM DA prior to Mn exposure. DA pre-treatment led to a significant leftward shift in the Mn dose-response survival curve (p,0.001) with a LD50 = 25 mM (Figure 3A), while 10 mM acute DA treatment alone did not affect the worms’ survival (Figure 3B). These results establish that exogenously applied DA and Mn can act synergistically in vivo to promote increased toxicity in the worm. Endogenous DA potentiates Mn toxicity In certain mutant backgrounds, such as vertebrate DAT mutants, DA levels were reported to be abnormally elevated due to a lack of DA clearance at the DAergic synapses [71]. Therefore, the effect of DAT-1 loss-of-function on Mn sensitivity may reflect increased endogenous levels of DA (Figure 4A). To test this hypothesis, we measured DA levels in the C. elegans dat-1(ok157) mutants. We found that dat-1(ok157) endogenous DA levels were significantly higher when compared with wild-type worms (Figure 4B). To confirm the role of endogenous DA in Mn toxicity, we tested the effect of endogenous DA depletion on Mn sensitivity using the cat-2(e1112) loss-of-function mutant, in which the tyrosine hydroxylase (TH) activity is abolished, resulting in the absence of DA synthesis (Figure 4A and 4B). cat-2(e1112) mutants exposed to Mn revealed significant hyper-resistance (p,0.001), with a LD50 = 95 mM (Figure 4C). Moreover, the e1112 deletion rescued the dat-1(ok157) hypersensitivity (LD50 = 9 mM, Figure 3A) and led to hyper-resistance to Mn (p,0.001) with a LD50 = 83 mM for cat-2(e1112);dat-1(ok157) worms (Figure 4C). The LD50 difference between cat-2(e1112) and cat-2(e1112);dat- 1(ok157) worm strains was not statistically significant (p.0.05), suggesting that the cat-2(e1112) effect takes place upstream of dat- 1(ok157) in the same pathway (Figure 4A). Finally, we supple- Figure 3. Sub-lethal dopamine pre-treatment sensitizes wild-type worms to Mn exposure. (A) Pre-treatment with 10 mM dopamine renders wild-type worms hypersensitive (p,0.001) to subsequent MnCl2 exposure, bringing the LD50 from 47 mM to 25 mM. (B) 10 mM L1 acute treatment does not affect wild-type worm survival at 24 h. doi:10.1371/journal.pgen.1001084.g003 PLoS Genetics | www.plosgenetics.org 5 August 2010 | Volume 6 | Issue 8 | e1001084