from cat-2(e1112);dat-1(ok157) without DA pre-exposure (Figure 4C). These data further confirm that endogenous DA level regulation plays a key role in Mn toxicity and that DA itself is required for the full extent of Mn toxicity. Extracellular DA, and not intracellular DA, is involved in Mn-induced lethality Next, we determined which step along the DAergic metabolic pathway contributes to the synergistic toxic effect of Mn. To address this issue, endogenous DA levels at the L1 stage were measured in a combination of deletion mutants (Figure 4A). First, we sought to determine if DA release was necessary for the DA- dependent Mn-induced lethality. Accordingly, cat-1(e1111) mu- tants in which the vesicular monoamine transporter 2 (VMAT2) is defective, were exposed to Mn (Figure 4B). This mutant is unable to pack DA in secretory vesicles, thus abolishing DA synaptic release, while still producing normal levels of DA, at least at the L1 stage (Figure 4A). Upon exposure to Mn, cat-1(e1111) mutants were more resistant to Mn-induced lethality than wild-type worms (p,0.001), with a LD50 = 108 mM (Figure 4D), which was statistically insignificant when compared to the cat-2(e1112) mutant LD50 (p.0.05). These data establish that synaptic DA release is necessary for DA-dependent Mn-induced lethality and that extracellular DA, and not presynaptic DA, mediates this effect. Furthermore, the data imply that blockage of extracellular DA receptor activity (and the ensuing increase in extracellular DA levels) should exacerbate Mn sensitivity. C. elegans expresses three DA receptors, namely DOP-1, DOP-2 and DOP-3 (Figure 4A). A triple knock-out was generated [78], and as expected, the dop- 2(vs105); dop-1(vs100) dop-3(vs106) mutant exhibited higher levels of DA (Figure 4B) and was hypersensitive (p,0.001 vs. wild-type) to Mn-induced lethality with a LD50 = 27 mM (Figure 4D). However, this mutant was not as sensitive to Mn as the dat- 1(ok157) mutant, probably because the DA clearance activity of the wild-type DAT-1 limited the extent of the extracellular DA accumulation in the dop-2(vs105); dop-1(vs100) dop-3(vs106) triple mutant. These results confirm that extracellular, and not intracellular DA is responsible for the DA-dependent Mn-induced lethality. DA-dependent Mn toxicity is associated with decreased longevity To further investigate the pathways involved in the DA- dependent Mn toxicity, and especially its long-term effects, we scored the survival rate of worms which survived acute Mn- exposure. For each strain tested, we picked healthy-looking young adults homogeneous in stage at 48 h post-exposure (for all strains tested these animals were representative of more than 90% of the surviving population), and disregarded sick-looking or develop- mentally delayed worms. Accordingly, all animals selected survived at least 4 days in adulthood, regardless of their genetic background. This bias was deemed necessary to ensure that any significant differences potentially observed between strains could not be attributed to early toxicity events. In accordance with previous reports [79], low doses of Mn (3 mM for 30 min) led to a minor (albeit insignificant) increase in the surviving wild-type lifespan from 14.5 days post-treatment (dpt) to 15 dpt (Figure 5A, upper and middle graphs). Higher doses of Mn (100 mM for 30 min) led to a decrease in lifespan from 14.5 dpt to 13.8 dpt (p,0.001, Figure 5A, bottom graph). dat-1(ok1057) mutants exhibited a significantly shorter lifespan compared with wild-type worms in the absence of Mn treatment (13.2 dpt, p,0.001), BLI-3/Dopamine-Dependent Mn-Induced Neurotoxicity which was further reduced (p,0.001) by both low- and high-dose acute Mn treatments (12 dpt and 11.7 dpt, respectively). Conversely, the lifespan of short-lived cat-2(e1112) mutants (12.3 dpt in absence of Mn, p,0.001) was significantly increased with increased Mn dosing (p,0.001, 14.6 dpt upon 3 mM and 14.8 dpt upon 100 mM acute Mn exposure). Moreover, the lifespan of the cat-2(e1112) mutants was significantly extended as compared to wild-type worms upon 100 mM Mn exposure (14.8 dpt vs 13.8 dpt, p,0.001), reaching untreated wild-type worm average lifespan (14.8 dpt vs 14.5 dpt). These observations made on worms, all of which survived for at least 5 days post-treatment, mirror the results obtained for lethality rates at 24 h post- treatment, indicating that the DA-dependent Mn toxicity observed upon acute Mn treatment has short- and long-term components, affecting both developmental (see also [66]) and aging processes. DA-dependent Mn-induced toxicity is associated with increased oxidative stress Toxicity mechanisms implicated in neurodegenerative diseases, such as PD and Alzheimer’s disease, involve oxidative stress [32,35,37,80,81,82,83]. In particular, DAergic cell loss in PD patients and experimental PD models invokes excessive ROS production [77,84,85,86,87,88]. Moreover, oxidizing metals such as copper, Mn and iron in their 2+ or 3+ states are known to be sources of ROS via the Fenton reaction [89]. In C. elegans, pre- treatment with anti-oxidants such as ebselen, affords protection against acutely-induced Mn toxicity (Avila and Aschner, unpublished data). To determine if oxidative stress plays a role in DA-dependent Mn toxicity in C. elegans, a double-pronged approach was undertaken. First, the presence of ROS in response to acute Mn treatment was determined with the fluorescent dye, 2979 dichlorodihydrofluorescein diacetate (H2DCF-DA). As shown in Figure 5B, Mn-treated wild-type worms showed a significant (p,0.05) increase in fluorescence at sub-lethal Mn doses (10 mM). Interestingly, dat-1(ok157) mutants showed a significant increase in fluorescence (p,0.01) after 1 mM exposure, whereas cat-2(e1112); dat-1(ok157) double mutants did not show any significant increase in fluorescence (p.0.05) upon exposure up to 30 mM Mn (Figure 5B). To confirm these results, we also used a more quantitative method involving the measurement of lipid peroxidation. Isoprostanes F2 (F2IP) and F3 (F3IP) are oxidation products of arachidonic acid (AA), which is released from membranes upon oxidative injury [90,91,92]. A new protocol was developed to extract and measure F2IP and F3IP from C. elegans. Corroborating the H2DCF-DA results (Figure 5B), dat-1(ok157) mutants exhibited significantly higher levels of F2IP (Figure 5C) and F3IP (data not shown) upon 1, 3 and 5 mM Mn exposure (p,0.05, p,0.001, p,0.05, respective- ly), as compared to wild-type worms, which showed a significant increase in F2IP content upon 5 and 10 mM Mn exposure (p,0.05 and p,0.001, respectively) (Figure 5C). The double mutant cat-2(e1112);dat-1(ok157) consistently displayed decreased F2IP levels between 1 and 5 mM Mn, with significantly lower levels at 3 mM (p,0.05), and exhibited significantly higher F2IP levels upon 10, 20 and 30 mM acute Mn exposure (p,0.05, p,0.01, p,0.001, respectively) (Figure 5C). Thus, to attain maximal levels of F2IP (corresponding to a 2-fold increase), higher doses of Mn were required in cat-2(e1112);dat-1(ok157) mutants compared to wild-type and in wild-type compared to dat- 1(ok157). Taken together, the H2DCF-DA and isoprostane measurements show that acute Mn treatments induce oxidative stress in C. elegans. In addition, the degree of oxidative stress depends on the extracellular DA content. These experiments PLoS Genetics | www.plosgenetics.org 7 August 2010 | Volume 6 | Issue 8 | e1001084