BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity Figure 1. Dopaminergic neurons specifically degenerate in a dose-dependent manner upon Mn acute exposure. (A) Mechanosensory termini of the CEP and ADE dopaminergic neurons in dat-1::GFP expressing worms display discontinuous GFP staining 24 h after 50 mM Mn acute exposure at L1, revealing their degeneration. (B) Adult CEP, ADE, PDE and male specific R5A, R7A and R9A pairs of neurons are also affected following 50 mM MnCl2 acute exposure at L1. (C) GABAergic (unc-47::GFP), cholinergic (unc-17::GFP) and chemosensory neurons (DiI) are not affected by Mn exposure at concentrations inducing dopaminergic neurodegeneration (50 mM here). (D) CEP and ADE degeneration upon Mn exposure is dependent on Mn concentration. White arrowheads indicate neuronal processes exhibiting abnormal discontinuous GFP signal. White stars indicate the position of degenerated neuron cell bodies. doi:10.1371/journal.pgen.1001084.g001 effect was specific to DAergic neurons, since it was not observed in GABAergic, cholinergic, glutamatergic (Figure 1C) or in other biogenic-amine systems (data not shown). Despite the direct exposure of chemosensory neurons to the Mn-containing solution, DiI staining in these neurons failed to reveal any degeneration (Figure 1C), confirming the specificity of the Mn- induced DAergic neurodegeneration. DAergic neurodegeneration requires the DA re-uptake transporter DAT-1 The selectivity of Mn-induced neurodegeneration to DAergic neurons suggests that some factor(s) specific to these neurons sensitize(s) them to Mn-induced toxicity. To determine which factor(s) may account for this effect, a candidate gene approach was employed, starting with a gene known to be specific to the DAergic neurons, namely, dat-1. DAT-1 is the C. elegans orthologue of the vertebrate DAT, which is a highly conserved member of a family of transporters involved in neurotransmitter clearance, including the GABA re-uptake transporters, the GATs, the serotonin re-uptake transporter, SERT, and the excitatory amino-acid transporters, the EAATs. DAT is specifically respon- sible for DA clearance at the synapse, removing excessive extracellular DA into presynaptic DAergic termini [67,68,69]. Accordingly, chemical inhibition of DAT or DAT loss-of-function leads to high extracellular DA levels [70,71]. In C. elegans, the dat- 1(ok157) loss-of-function mutant displays a swimming-induced PLoS Genetics | www.plosgenetics.org 3 August 2010 | Volume 6 | Issue 8 | e1001084