Author Summary In Parkinson’s disease (PD), motor neurons that produce dopamine degenerate, leading to a characteristic syn- drome including tremor, rigidity, and bradykinesia. The mechanisms leading to PD have been under intense investigation, identifying hereditary mutations responsible for about 8% of the cases. However, multiple environ- mental factors contribute to PD; and, amongst those, manganese (Mn) exposure from pesticides, industrial fumes, and gasoline additives has been robustly associated with PD. To gain insights into processes leading to the specific degeneration of dopaminergic neurons, we used a simple animal model, the nematode Caenorhabditis elegans, which, upon Mn exposure, recapitulates key molecular processes known to be involved in PD. Combining biochemistry and genetics, we demonstrate that dopamine secreted by the neurons and not intracel- lular dopamine is directly involved in the generation of toxic reactive oxygen species. We identify two essential mediators of this dopamine-dependent effect which are an extracellularly active enzyme called dual-oxidase and the dopamine re-uptake transporter. We also reveal that a transcription factor which is strongly expressed in two neurons involved in the regulation of aging is a powerful modulator of the dopamine-dependent toxicity. Our study establishes novel evidence of the link among PD, aging, and oxidative stress within the context of exposure to Mn. Appraisal of the literature strongly suggests that in addition to targeting similar brain areas and causing similar clinical syndromes, DAergic neurodegeneration associated with PD or PD-mimicking drugs (6-hydroxydopamine/6-OHDA, 1-methyl-4- phenylpyridium/MPP+, rotenone, paraquat) and Mn neurotoxic- ity share multiple common effector mechanisms, namely mito- chondrial dysfunction, ATP depletion, aberrant signal transduc- tion, oxidative stress, protein aggregation and the activation of cell death pathways [32]. Damage to DAergic nigral neurons that is induced by MPP+ and rotenone involves oxidative stress [33]. Oxidative damage also plays a significant role in 6-OHDA- induced DAergic neuronal cell death. In cell cultures, hydrogen peroxide (H2O2), superoxide ions and hydroxyl radicals [34] generated by the non-enzymatic breakdown of 6-OHDA and the direct inhibition of complex-I activity, lead to lipid peroxidation, protein denaturation and a decrease in glutathione (GSH), all hallmark features of post-mortem PD [35,36,37]. Intrastriatal Mn injections result in the loss of DAergic neurons, a process in which oxidative stress plays a significant role [38,39,40], resembling toxicity caused by the mitochondrial poisons, aminooxyacetic acid and MPP+ [41]. Similarly to mitochondrial inhibitors such as MPP+ [42], Mn increases in vivo synaptic glutamate concentrations, which leads to excitotoxic and oxidative injury [43] and interfers with ATP synthesis [44]. Analogous to MPP+ and 6-OHDA, Mn elevates intracellular H2O2 and related peroxides [45] and reduces tyrosine hydroxylase (TH) activity and intracellular antioxidant levels (GSH, thiols, catalase) in DAergic neurons [43,46,47,48]. Intracellular Mn2+ inhibits the mitochondrial complex-I, a feature inherent to PD and its experimental models (MPP+, 6-OHDA, rotenone, paraquat) [43]. A link between mitochondrial impairment, oxidative stress and increased a-synuclein aggregation is well documented for Mn and in various models of PD [37,43,49,50,51]. Studies have also confirmed that treatment with Mn in a pre-parkinsonian state (6- OHDA) significantly exacerbates neurobehavioral impairment in the rat, not only suggesting that Mn exposure may increase the risk BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity of injury in subpopulations that are in a pre-parkinsonism state, but also pointing to the convergence of signaling pathways that lead to such injury [52]. MPP+ and 6-OHDA exposures as well as wild-type or mutant a- synuclein overexpression cause specific DAergic neurodegenera- tion in the worm [53,54,55,56], a process which involves ATP depletion and oxidative stress [36,57,58], analogous to vertebrate models of PD. It was further confirmed that C. elegans orthologues of PD-associated genes play a role in a-synuclein toxicity and DAergic neurodegeneration. Additionally, conserved genetic networks were identified in C. elegans that potentiate or protect against a-synuclein toxicity, such as the torsin pathway [36,59,60,61,62,63,64]. A link between a-synuclein and Mn toxicity was also demonstrated in the worm [59,65]. Furthermore, we previously established that Mn uptake and toxicity pathways in C. elegans relate to those described in vertebrates, which involves the NRAMP/DMT family of metal transporters and leads to defects in the developmental and excretory systems [66]. Here we show that at a sub-lethal range of concentrations, acute Mn exposure leads to a specific and dose-dependent neurodegenera- tion of all C. elegans DAergic neurons, while sparing other neurotransmitter systems, findings that corroborate the specificity of DAergic sensitivity shared with vertebrate models. We investigated the causes of this DAergic specificity, and we demonstrated that endogenous extracellular, but not intracellular DA potentiates Mn toxicity and that Mn-induced neurodegener- ation requires the DAergic neuron-specific dopamine re-uptake transporter, DAT-1. We also found that Mn toxicity in the worm is associated with increased reactive-oxygen species (ROS), lipid peroxidation and lifespan reduction, all of which were dependent on extracellular DA concentrations. Additionally, we observed a relocation of the oxidant-responsive transcription factor, SKN-1, in ASI nuclei upon Mn exposure, whereas SKN-1 overexpression afforded protection against Mn-induced toxicity. Finally, we identified the NADPH dual-oxidase, BLI-3, as a key mediator of the DA-dependency of Mn toxicity, suggesting that BLI-3 potentiates the formation of ROS from DA-derived species obtained through the reaction of divalent Mn and extracellular DA. Results C. elegans DAergic neurons degenerate in a specific and concentration-dependent manner upon exposure to Mn We first ascertained the suitability of Mn-exposed C. elegans as an in vivo model for manganism and PD and examined whether features inherent to mammalian DAergic neurodegeneration can be convincingly recapitulated in this model. We took advantage of the BY200 strain, which expresses the green fluorescent protein (GFP) under the control of the DAergic-specific dopamine re-uptake transporter 1 promoter, dat-1::GFP(vtIs1) (Figure 1A). After acute exposure to Mn, a dose-dependent neurodegeneration was observed in all DAergic neurons, namely the 4 CEP, 2 ADE, 2 PDE and the male specific R5A, R7A and R9A pairs of neurons (Figure 1A and 1B). Typically, the primary defects were observed in neuron extensions, such as CEP mechanosensory processes, resulting in discontinued and punc- tuated GFP labeling (Figure 1A, 1B, 1D, arrowheads). With an increased dose (Figure 1D, upper to lower panels) or longer exposure to Mn (data not shown), these defects were exacerbated, leading to shortening or disappearance of the neuronal extensions (Figure 1D, lower panels) and eventually neuronal death as revealed by the shrinkage of the cell body, and ultimately, complete loss of GFP (data not shown, Figure 2A left panel). This PLoS Genetics | www.plosgenetics.org 2 August 2010 | Volume 6 | Issue 8 | e1001084