BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity Figure 8. Recapitulative scheme. Manganese taken up by the epithelial DMT1-like SMF transporters is released in the body cavity (possibly by ferroportins), where it meets extracellular dopamine released from the dopaminergic synapses. The dual-oxidase BLI-3 is likely involved in the generation of oxidized dopamine-derived species, which can be taken up by DAT-1 leading to dopaminergic neurodegeneration, or are further increasing overall oxidative stress, associated with larval lethality and adult lifespan reduction. The ASI neurons in which SKN-1 nuclear relocation was observed could be involved in the orchestration of the overall antioxidant response. Dashed arrows highlight the pathways supported by this study. ‘‘SMFs?’’: SMF transporters may be involved in direct Mn uptake in DAergic neurons according to earlier studies [65,66]. doi:10.1371/journal.pgen.1001084.g008 and dose-dependency of the DAergic neurodegeneration [110,111]; the involvement of DAT [112,113,114]; the synergy between DA and Mn [115]; and the associated oxidative stress [116,117,118]. As many divalent metallic cations are able to oxidize biogenic amines (amongst which DA and serotonin) in vitro via the Fenton’s reaction, we carried out two control experiments to ascertain the specificity of the relationship between DA and Mn in vivo. Unlike dat-1 mutation, deletion of the serotonin transporter (mod-5 in C. elegans) did not affect Mn-induced lethality (LD50 = 45 mM, Figure S1), and DA-depleted cat-2(e1112);dat-1(ok157) mutants revealed greater susceptibility to iron-induced ROS production (p,0.05) than wild-type worms (Figure S2), supporting the fact that in vivo, metal-induced DA-dependent toxicity is specific to Mn. An additional novelty resides in the finding of the early and central role played by endogenous extracellular DA and the NADPH dual-oxidase in Mn-induced toxicity (Figure 4, Figure 5, and Figure 7). The centrality of extracellular DA in Mn-induced toxicity was further exemplified by the level of resistance to Mn conferred by mutations in cat-1 (LD50 = 108 mM) and cat-2 (LD50 = 95 mM), which approach those conferred by deletion of the Mn transporter, smf-3 (LD50 = 126 mM) [66]. Moreover, Mn in its normal toxic range had a strong beneficial effect on the lifespan of DA-depleted cat-2(e1112) mutants (Figure 5A), further confirming that the DA-dependent component of Mn-induced toxicity accounts for a significant share of the overall toxicity. Importantly, the hypersensitivity to Mn of the DA receptor triple- knockout (Figure 4D), shows that Mn-induced neurodegeneration does not require DA receptors. Combined with the absence of post-synaptic neurodegeneration in dop-1::GFP and dop-3::RFP- expressing worms upon Mn exposure (data not shown) and despite the excitotoxic potential of other DA-related compounds, such as L-DOPA and 6-OHDA [119], the results establish that DA- dependent Mn toxicity does not involve excitotoxicity, in contrary to the glutamate-induced neurodegeneration involved in amyo- trophic lateral sclerosis (ALS) [120]. Together, these results also provide a novel explanation for the requirement of DAT in the DAergic neurodegeneration in vivo. Rather than being responsible for cytosolic DA accumulation followed by downstream ROS production, which was considered as the reason for the sensitivity of DAergic neurons to PD-mimicking drugs (MPP+, 6-OHDA) [56,114,121,122], DAT may facilitate the transport of extra- cellulary oxidized DA-derived species into DAergic neurons. In man and rat, subchronic Mn exposure has been shown to reduce DAT expression levels [123,124]. Although it is unclear if it reflected a decline of DAergic neuron activity or a specific down- regulation of DAT, Mn interference with DAT activity should be further investigated. The importance of the NADPH dual-oxidase, BLI-3, in the DA-dependent Mn toxicity in C. elegans corroborates the up-regulation of its mammalian orthologue in PD patients and MPP+-exposed mice, as well as the protective effect conferred by its loss-of-function against oxidative stress and DAergic neurode- generation in MPP+-exposed mutant mice [86]. Finally, DA- dependent Mn-induced toxicity has both short and long-term components as revealed by survival rate and oxidative stress measurements at 24 h (Figure 4, Figure 5B and 5C) and by lifespan data (Figure 5A). The protective effect afforded by SKN-1 upregulation upon Mn exposure and its nuclear relocation in ASI neurons (Figure 6) emphasizes the influence of acute Mn treatments on aging. ASI neurons which strongly express SKN- 1, were shown to play a key role in lifespan modulation through the well-studied diet-restriction pathway [125]. Hence, further examination of Mn-induced toxicity and lifespan reduction in ASI neuron-ablated worms, skn-1(RNAi) worms and mutants of the diet-restriction pathway may provide essential insights in the relationship between aging and PD, as well as on the role of specific brain area (corresponding to the C. elegans ASI neurons) on aging and PD. Despite the absence of an obvious PD-like behavior in the worm, DAergic neurodegeneration in C. elegans induced either by PD-drug treatment (6-OHDA, DA, MPP+) or by Mn involves the same key genes and molecular pathways as in vertebrates [32,36,54,59,60,64,65,66,126,127,128], has both short-term and long-term components and therefore represents a powerful model PLoS Genetics | www.plosgenetics.org 11 August 2010 | Volume 6 | Issue 8 | e1001084