regulator of antioxidant response from man to worm [94,99], and its impact on lifespan could as well be due to its ability to reduce oxidative stress naturally occurring with age, without involving caloric restriction. Given the impact of Mn exposure and DA metabolism on oxidative stress (Figure 5B and 5C), SKN-1 overexpression may improve Mn-exposed worm survival merely by compensating for the excessive ROS produced when extracellular DA levels are high and/or when Mn exposure reaches toxicity levels. Interestingly, the survival dose-response curve obtained with a non-null mutation (ok2315) in a mixed population of heterozygous and homozygous animals (Figure 6A) showed that even for very low doses of Mn (0.001 and 0.01 mM), a noticeable fraction of the worms (about 15–20%) died at 24 h post treatment compared to untreated animals, making it difficult to fit the experimental data with a simple sigmoid dose-response curve. As it is a mixed population, it is likely that mostly homozygous mutants died at those lowest doses, suggesting that their LD50 could be in the submillimolar range, making skn-1(ok2315) worms the most Mn-sensitive mutants tested so far. This requires direct confirmation, but it further supports an essential role for SKN-1 in regulating even slight changes in oxidative stress levels. Second, our lifespan experiments showed that both dat-1 and cat-2 mutants are short-lived in control conditions (no published literature could be identified reporting on the lifespan of these mutants) compared to wild-type worms (Figure 5A). According to our ROS and isoprostane measurements (Figure 5B and 5C), dat-1 mutants show increased oxidative stress, which is known to worsen with age; therefore explaining their shorter lifespan. Conversely, cat-2 mutants seemed much less affected by Mn-induced oxidative stress, as Mn treatment did not lead to detectable increase in ROS and only led to a significant increase in isoprostane levels above 10 mM of Mn exposure. Moreover, high Mn exposure was able to rescue the shorter lifespan of cat-2 mutants (Figure 5A). Mn is naturally required for the antioxidant activities of several enzymes, such as catalase and superoxide dismutase, which activities are critical in the aging brain [153,154,155]. Mn also has bactericidal and fungicidal [156] properties that are exploited in pesticides, such as Maneb. Several hypothesis can be formulated: (1) cat-2 mutants may take up less Mn than wild-type worms, possibly below optimal levels, (2) DA depletion in cat-2 mutants could make them feed improperly and somehow normally toxic Mn doses would restore the metabolic balance in those worms, (3) cat-2 worms may be prone to infections and high Mn exposure would help the worms cope with a weak immunity. The first point could involve DA as a regulator of feeding behavior. This aspect can be tested by measuring Mn levels, the basal slowing response [157], sharp angle turns [158] and the pharyngeal pumping rate of dat-1, cat-2, cat-2;dat-1 and wild-type animals upon Mn exposure. Those tests would also allow functional characterization of the DAergic circuit of Mn-exposed worms. The second hypothesis involves energy depletion as a cause of shortened lifespan, whereas the stress of Mn exposure would restrict energy expenditure by inhibiting the Insulin/IGF-1 pathway, possibly involving SKN-1 activation. In this case, Mn intake may not be different from other worms. Mn level measurements, Nile red and oil red O [159] staining to monitor fat stores, as well as RT-PCR and western- blots to measure daf-2, akt-1/-2, sgk-1, daf-16 and skn-1 expression levels would allow to test this idea. The third hypothesis implies that DA-derived ROS naturally play a role in the worm’s immunity and that a high Mn dose compensates for their absence in cat-2 mutants. The reasoning behind this idea is detailed in the next paragraph. This could be easily tested by comparing the resistance to infections of wild-type, dat-1, cat-2, cat-2;dat-1 and bli- 3 mutants in presence or absence of Mn. BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity Mn, DA, BLI-3: a link between immunity and neurodegeneration? Our data show that DA and BLI-3-dependent ROS production is triggered or amplified by Mn, which was shown to be taken up by the NRAMP/DMT orthologues, smf-1, -2 and -3 [66]. Recently, bli-3 and BLI-3-generated ROS have been implicated in C. elegans defense against bacteria and fungi [105,106]. The NRAMP/DMT family of metal transporters has a well-established role in innate immunity in vertebrates [160,161,162,163]. In C. elegans, NRAMP/DMT orthologue deletion mutants were found to be hypersensitive to Staphyloccocus aureus infection, which was rescued by Mn feeding [164]. Involvement of BLI-3-generated ROS, Mn and NRAMP/DMT in the defense against pathogens in C. elegans, as well as in DA-dependent neurodegeneration, raises the question of a link between immunity and DAergic neurode- generation. The finding that DA and Mn act synergistically in a BLI-3-dependent ROS production pathway suggests that DA may play a direct role in the worm’s immunity. If this mechanism is conserved in vertebrates, perhaps as a relic of a primitive immune system, it could bear interesting implications for brain physiology. For instance, DA-derived ROS could help fight infections when the blood-brain barrier (BBB) is compromised. On the other hand, they could also injure the brain and more specifically the DAT- expressing DAergic neurons. Infections and inflammation have long been suspected to play a role in the etiology of PD, as various infections have been associated with cases of PD [165,166,167,168,169,170,171,172]. The identification of a DA- derived ROS production mechanism implicating dual oxidases may hold some clues for the understanding of those associations. Summary This study confirms the conservation across the animal kingdom of molecular pathways involved in manganism and PD, provides the grounds for further biochemical and genetic investigations using the C. elegans model to tackle the complex issue of environment-gene interactions in age-related DAergic neurode- generative disorders, and unravels the essential role of extracellular DA oxidation and the NADPH dual-oxidase, BLI-3, upstream of DAT-1 requirement in the neurodegeneration pathway. The data also point to a genetic link with distinct and more general aging processes, such as the diet-restriction pathway through the involvement of SKN-1, and with an innate immunity genetic network involving metal-content regulation via the SMF trans- porters and oxidative defence mechanisms via BLI-3 in C. elegans. Materials and Methods C. elegans strains and handling of the worms C. elegans strains were handled and maintained at 20uC as previously described [173]. The following strains were used: N2 (+); BY200, dat-1::GFP(vtIs1) V; BZ555, dat-1::GFP(egIs1); VH15, glr-1::GFP(rhIS4) III; LX929, unc-17::GFP(vsIs48); EG1285, lin- 15B(n765) unc-47::GFP(oxIs12) X; DA1240, adIs1240[lin-15((+) eat- 4::GFP) lin-15B(n765) X; LX734, dop-2(vs105) V; dop-1(vs100) dop- 3(vs106) X; LX831, lin-15B(n765) X; DOP-1::GFP(vsIs28) dop- 3::RFP (vsIs33); RM2702, dat-1(ok157) III; MT9772, mod-5(n3314) I; CB1111, cat-1(e1111) X; CB1112, cat-2(e1112) II; CB767, bli- 3(e767) I; BY602, cat-2(e1112) II; dat-1(ok157) III; BY645, dat- 1(ok157) III; dat-1::GFP(vtIs1) V; VC1772, skn-1(ok2315) IV;nT1[- qIs51](IV;V). All strains were provided by the Caenorhabditis Genetic Center (CGC, Minnesota), except for the BY602 and BY645 strains, which were generously provided by Randy Blakely (Vanderbilt University Medical Center, TN, USA). PLoS Genetics | www.plosgenetics.org 13 August 2010 | Volume 6 | Issue 8 | e1001084