to investigate genetic and environmental causes of PD and manganism. To further demonstrate the relevance of our findings to vertebrate physiology, it would be interesting to test (1) whether co-treatments of DA and Mn lead to the same synergy in DAergic neurodegeneration in rodents; (2) whether such synergy is dependent on DAT; and (3) whether tyrosine hydroxylase (TH/ CAT-2) and VMAT inhibition affords neuroprotection. Establish- ing the contribution of vertebrate NADPH dual-oxidases to the oxidation state of DA in the absence or presence of Mn would also be important. The present study also revealed DA-dependent mechanisms of Mn-induced toxicity by focusing on a whole- organism approach, while acknowledging that further investiga- tion should also focus specifically on DAergic neuron physiology. For instance, determining if upon Mn exposure, cat-2, cat-1 and cat-2;dat-1 mutants display less or no neurodegeneration and if DA supplementation can reverse this effect, would allow clearer understanding of the role of DAT, and intracellular and extracellular DA in the demise of the DAergic system. It would also be important to develop full-length GFP-tagged protein markers of the DAergic neurons as the dat-1::GFP probe can only reveal relatively advanced stages of neuronal decay, and not more subtle functional changes. This point is exemplified by the observation of SKN-1::GFP relocation in ASI neurons (Figure 6), which would have been missed if only a transcriptional GFP reporter approach had been used. Finally, exploring the effect of aging and diet-restriction modulated by the ASI neurons and the Nrf2-like transcription factor, SKN-1, would enhance our understanding of the pathophysiology and the progressive nature of PD. Further developments and implications for the treatment of DAergic neurodegeneration Given the similarities between Mn toxicity in C. elegans and in vertebrates [66] as well as the great conservation of most PD- related genes involved in DAergic neurodegeneration in rodents and humans [32,36,59,60,64,129], it is likely that the DA- dependent toxicity revealed herein plays an important role in diseases associated with DAergic neuron loss in mammals. The importance of extracellular DA levels in the toxicity mechanisms described here, if corroborated in vertebrates, could bear important implications for the treatment of DAergic neurodegen- eration, as modulating extracellular vs. intracellular DA levels requires different strategies. For instance, L-DOPA is prescribed in PD and manganism patients to treat the tremor and bradykinesia arising from the loss of DAergic activity. If L-DOPA, like DA, led to the excessive generation of oxidized reactive species in vivo, L- DOPA treatment, although compensating for the DA loss, could accelerate or exacerbate the DAergic neurodegeneration over a longer term. If the involvement of dual-oxidases in DA oxidation was confirmed in vertebrates and if L-DOPA was shown to be easily oxidized by dual-oxidases, it would be important to design alternative DA analogues, which maintain high affinity for DA receptors and DAT but are poor substrates for the dual-oxidases. Another strategy to limit the extent of the DAergic neurodegen- eration could be the direct inhibition of the dual-oxidases. Finally, the protective effect of SKN-1 overexpression on Mn-induced toxicity (Figure 6A), which mirrors the neuroprotection afforded by astrocytic overexpression of Nrf2 in mice [130], suggests that promoting Nrf2 activity may be beneficial in limiting Mn-induced toxicity [131,132]. Such an approach would be particularly relevant to welding and smelting activities in which workers exposed to metal fumes have an increased risk of developing parkinsonian syndromes [28,133,134,135,136]. BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity Nature of the DA-derived oxidized species and oxidative- stress pathways involved in PD The current literature provides a conceptual framework for addressing the synergistic mechanism of Mn and DA in DAergic neurodegeneration [76,137,138,139,140,141]. Accordingly, Mn may enter DAergic neurons via DAT as a complex with DA-derivatives, such as dopaminochrome [138,141,142,143,144,145,146,147]. This hypothesis provides explanations both for the specificity of Mn toxicity towards DAergic neurons as well as for the synergistic toxicity of extracellular DA and Mn, while remaining consistent with studies that report a significant Mn uptake by the DAergic neurons [148,149,150,151]. Purification and identification of the DA-derived reactive species (using HPLC-ED [152]) are essential for proving these suggestions. Further quantification of those DA- derived species in Mn-, MPP+-, 6-OHDA- and DA-exposed worms or in the rodent brain and cerebrospinal fluid should help identify the key toxic species in DAergic neurodegeneration as well as point out candidate enzymes possibly involved in PD. However, in biochemical approaches, the timing of extraction is critical to robustly detecting and measuring those species. This concern is exemplified by our oxidative stress marker measure- ments in wild-type and dat-1(ok157) mutant worms (Figure 5B and 5C). Hence, above the Mn dose reaching the maximum increase in ROS or F2IP levels, higher Mn doses led to lower oxidative stress marker levels suggesting a decrease in oxidative stress, while the lethality rate at 24 h and lifespan results suggest otherwise (Figure 4, Figure 5A). It is unlikely that oxidative stress decreased past the dose showing the maximum increase in oxidative stress markers. Downstream conversion or degradation of ROS or F2IP associated with a worsening of the condition of the worm may be responsible for this effect. Using genetic backgrounds that stop or delay the oxidative cascade at different steps of the pathway would allow the accumulation of specific ROS and increase their detection. Hence, Mn-resistant C. elegans mutants exposed to medium- to high-dose treatments (in the 20 to 100 mM range) could be very helpful in identifying the early steps of the oxidative cascade(s) leading to DAergic neurodegeneration and could yield new molecular targets for PD treatment. This study identifies a few of the potential candidates (bli-3, SKN-1 overexpressing and maybe cat-1 mutant worms, Figure 4 and Figure 6), but genetic screens to isolate new Mn-resistant mutants would provide powerful means for further investigation. Role of Mn and DA in C. elegans longevity In the course of this study, we came across several indications of the influence of the DA-dependent and Mn-induced toxicity on C. elegans longevity, which is unlikely to be a direct consequence of DAergic neurodegeneration, but rather a concomitant effect (dat- 1(ok157) mutants did not show DAergic neurodegeneration, whereas their lifespan decreased upon Mn exposure). First, the fact that SKN-1 protects against Mn-induced toxicity and modifies its nuclear localization pattern upon Mn exposure in ASI neurons (Figure 6), provides a genetic link with the Insulin/ IGF-1 and caloric restriction pathway known to be involved in the modulation of lifespan in C. elegans [97]. The nuclear relocation of SKN-1 in ASI neurons in a punctuate pattern is suggestive of binding of this transcription factor to specific chromosomal regions, likely corresponding to the loci of its downstream targets. Chromatine immunoprecipitation experiments could be used to identify those loci, while generation of transgenic worms expressing SKN-1::GFP and tagged sequences corresponding to candidate gene (for instance the superoxide dismutase genes) regulatory sequences would allow immuno-colocalization of SKN-1 and its targets. Nrf2/SKN-1 is known to be a key PLoS Genetics | www.plosgenetics.org 12 August 2010 | Volume 6 | Issue 8 | e1001084