Extracellular Dopamine Potentiates Mn-Induced Oxidative Stress, Lifespan Reduction, and Dopaminergic Neurodegeneration in a BLI-3–Dependent Manner in Caenorhabditis elegans Alexandre Benedetto1,2,3, Catherine Au1,4, Daiana Silva Avila1,2, Dejan Milatovic1,4, Michael Aschner1,2,4* 1 Department of Pediatrics, Vanderbilt University, Nashville, Tennessee, United States of America, 2 Children’s Hospital, Vanderbilt University, Nashville, Tennessee, United States of America, 3 London Centre for Nanotechnology, University College London, London, United Kingdom, 4 Center for Molecular Toxicology, Vanderbilt University, Nashville, Tennessee, United States of America Abstract Parkinson’s disease (PD)-mimicking drugs and pesticides, and more recently PD-associated gene mutations, have been studied in cell cultures and mammalian models to decipher the molecular basis of PD. Thus far, a dozen of genes have been identified that are responsible for inherited PD. However they only account for about 8% of PD cases, most of the cases likely involving environmental contributions. Environmental manganese (Mn) exposure represents an established risk factor for PD occurrence, and both PD and Mn-intoxicated patients display a characteristic extrapyramidal syndrome primarily involving dopaminergic (DAergic) neurodegeneration with shared common molecular mechanisms. To better understand the specificity of DAergic neurodegeneration, we studied Mn toxicity in vivo in Caenorhabditis elegans. Combining genetics and biochemical assays, we established that extracellular, and not intracellular, dopamine (DA) is responsible for Mn- induced DAergic neurodegeneration and that this process (1) requires functional DA-reuptake transporter (DAT-1) and (2) is associated with oxidative stress and lifespan reduction. Overexpression of the anti-oxidant transcription factor, SKN-1, affords protection against Mn toxicity, while the DA-dependency of Mn toxicity requires the NADPH dual-oxidase BLI-3. These results suggest that in vivo BLI-3 activity promotes the conversion of extracellular DA into toxic reactive species, which, in turn, can be taken up by DAT-1 in DAergic neurons, thus leading to oxidative stress and cell degeneration. Citation: Benedetto A, Au C, Avila DS, Milatovic D, Aschner M (2010) Extracellular Dopamine Potentiates Mn-Induced Oxidative Stress, Lifespan Reduction, and Dopaminergic Neurodegeneration in a BLI-3–Dependent Manner in Caenorhabditis elegans. PLoS Genet 6(8): e1001084. doi:10.1371/journal.pgen.1001084 Editor: Kaveh Ashrafi, University of California San Francisco, United States of America Received February 16, 2010; Accepted July 22, 2010; Published August 26, 2010 Copyright: 2010 Benedetto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH R01 ES10563 to MA. The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: michael.aschner@vanderbilt.edu Introduction Mn is the twelfth most prevalent natural element in the Earth’s crust [1] and is an essential transition metal required for normal growth, development and cellular homeostasis [2,3]. It acts as a cofactor for multiple enzymes (Mn superoxide dismutase, pyruvate carboxylase, arginase, and glutamine synthase) [4,5,6,7,8] and can substitute for magnesium (Mg) in many enzymatic reactions catalyzed by kinases. Although dietary Mn intake by the gastrointestinal tract (GIT) and excretion via the bile are tightly regulated [9], inhalation of toxic concentrations of Mn can lead to nasal and pulmonary inflammation, renal dysfunction and neurodegeneration [7,10,11,12]. A recent study also suggests that high levels of Mn in drinking water (.300 mg/liter) are associated with reduced intellectual function in children [13]. Mn mining, steel manufacturing and welding represent occupational exposures linked to increased risk for parkinsonian syndrome [14,15]. In addition, Mn is used in other industrial and agricultural applications. Fungicides, such as Maneb or Mancozeb, increase the risk of environmental Mn exposure in agricultural workers [16]. An organic Mn compound, methylcyclopentadienyl Mn tricarbonyl (MMT), used as an octane booster or anti-knock agent in gasoline, has also been shown to cause adverse health effects [17,18,19,20]. Exposure to high levels of Mn in occupational or environmental settings or disease conditions (hepatic encephalopathy) [21] is accompanied by Mn accumulation in specific brain regions that are highly sensitive to oxidative injury, namely the substantia nigra (SN), globus pallidus (GP) and striatum [22]. Excessive Mn deposition in these regions leads to dopaminergic (DAergic) neuronal loss accompanied by an extrapyramidal syndrome referred to as manganism. Manganism patients exhibit rigidity, tremor, dystonic movements and bradykinesia, all of which are also characteristic features of Parkinson’s disease (PD) [23,24,25]. Exposure to Mn also represents a risk factor for PD [26,27,28]. Indeed, the strongest correlation between any type of environmental exposure and PD is noted in Mn- exposed human cohorts [29], and occupational exposure to Mn for .20 years or combined long-term exposures to Mn and Al (.30 years) are associated with an increased occurrence of PD [30]. Parkinsonism in welders (vs. non-welders) is clinically distinguishable only by age of onset (46 vs. 63 years, respectively) [28], and the prevalence of PD is higher among welders as compared with age- standardized individuals in the general population [31]. PLoS Genetics | www.plosgenetics.org 1 August 2010 | Volume 6 | Issue 8 | e1001084