Neurotoxicity of the Parkinson Disease-Associated Pesticide Ziram Is Synuclein-Dependent in Zebrafish Embryos

Abstract

Background: Exposure to the commonly used dithiocarbamate (DTC) pesticides is associated with an increased risk of developing Parkinson disease (PD), although the mechanisms by which they exert their toxicity are not completely understood.

Objective: We studied the mechanisms of ziram's (a DTC fungicide) neurotoxicity in vivo.

Methods: Zebrafish (ZF) embryos were utilized to determine ziram's effects on behavior, neuronal toxicity, and the role of synuclein in its toxicity.

Results: Nanomolar-range concentrations of ziram caused selective loss of dopaminergic (DA) neurons and impaired swimming behavior. Because ziram increases α-synuclein (α-syn) concentrations in rat primary neuronal cultures, we investigated the effect of ziram on ZF γ-synuclein 1 (γ1). ZF express 3 synuclein isoforms, and ZF γ1 appears to be the closest functional homologue to α-syn. We found that recombinant ZF γ1 formed fibrils in vitro, and overexpression of ZF γ1 in ZF embryos led to the formation of neuronal aggregates and neurotoxicity in a manner similar to that of α-syn. Importantly, knockdown of ZF γ1 with morpholinos and disruption of oligomers with the molecular tweezer CLR01 prevented ziram's DA toxicity.

Conclusions: These data show that ziram is selectively toxic to DA neurons in vivo, and this toxicity is synuclein-dependent. These findings have important implications for understanding the mechanisms by which pesticides may cause PD. Citation: Lulla A, Barnhill L, Bitan G, Ivanova MI, Nguyen B, O'Donnell K, Stahl MC, Yamashiro C, Klärner FG, Schrader T, Sagasti A, Bronstein JM. 2016. Neurotoxicity of the Parkinson disease-associated pesticide ziram is synuclein-dependent in zebrafish embryos. Environ Health Perspect 124:1766-1775; http://dx.doi.org/10.1289/EHP141.

Figure 1

Ziram is selectively toxic to dopaminergic neurons. VMAT2 zebrafish (ZF) embryos were exposed to vehicle (A) and 50 nM ziram (B) at 24 hour post- fertilization (hpf). Neuronal counts for the telencephalic (TC) and diencephalic (DC) clusters were conducted at 5 days post-fertilization (dpf) (n = 5). A 30% decrease in telencephalic neurons (C) and a 39% decrease in diencephalic neurons (D) were observed in ziram-treated fish. Compared with controls (E), ziram’s toxicity did not extend to Rohon-Beard neurons (5 dpf, n = 7) after exposure to 50 nM ziram (F,G). Raphe nuclei neurons are also labeled in VMAT2:GFP fish (serotonergic neurons), and compared with controls (H), they were unaffected by ziram exposure (I). Raphe nuclei neuron counts are shown in (J). Scale bar = 500 μm. *, p < 0.05; **, p < 0.01. Two-tailed Student’s t-test.

Figure 2

Ziram causes alterations in zebrafish (ZF) motor behavior. Movement was tracked for larval ZF at 7 days post-fertilization (dpf) (n = 24). Distances > 2 mm were tracked under an alternating light (yellow)/dark (gray) cycle (A). A 24.7% decrease (p < 0.05) in distance traveled during periods of dark was observed in ziram-treated ZF relative to vehicle-treated ZF (B), but no significant difference was observed during periods of light (C). A similar pattern of swimming, less in the dark but no difference in the light, was measured when ZF were treated with the dopamine antagonist haloperidol (B,C). The dopamine agonist apomorphine (Apo) increased swimming to a similar degree in the ziram-treated and control ZF, suggesting that the postsynaptic dopaminergic system remained intact. No significant difference in motor behavior was observed for ziram + apomorphine versus apomorphine alone for light or dark conditions. All treatment groups were compared with vehicle controls for statistical analysis. *, p < 0.05; ***, p < 0.001; ****p < 0.0001; one-way analysis of variance (ANOVA).

Figure 3

ZF γ1 synuclein aggregates and forms fibrils similarly to human α-synuclein (α-syn). Recombinant human α-syn (hSyn) or ZF γ1 (both 150 μM) were incubated over a 66-hr period, and thioflavin T fluorescence was monitored (A). The morphology of hSyn (B) and ZF γ1 (C) was examined using transmission electron microscopy (TEM) at the end of the aggregation reaction (scale bar = 0.2 μm).

Figure 4

Overexpression of ZF γ1 in vivo causes intracellular aggregation of synuclein. Embryos were injected with constructs for HuC-T2A-DsRed (A) or HuC-γ1-T2A-DsRed (B); embryos shown at 2 days post-fertilization (dpf) (scale bar = 100 μm). Sectioned embryos were stained with a primary antibody for ZF γ1. HuC-DsRed–injected (C) embryos did not have intracellular ZF γ1 aggregates, whereas embryos injected with HuC-ZFγ1 (D) were found to have intracellular ZF γ1 aggregates (blue arrow) (scale bar = 10 μm). To determine if ZF γ1 formed β-pleated sheets in vivo, embryos injected with HuC-DsRed and HuC-γ1-T2A-DsRed were stained with thioflavin S (ThS) (scale bar = 10 μm). Only neurons overexpressing ZF γ1 (f) were ThS-positive (E).

Figure 5

Ziram decreases ZF γ1 levels. Embryos were treated with ziram or vehicle, and ZF γ1 levels were determined by Western blot analysis at 5 days post-fertilization (dpf) (normalized to tubulin). A 69.3% decrease in band density for ZF γ1 was observed for samples treated with 50 nM ziram (A,B, n = 4). *, p < .05. Two-tailed Student’s t-test.

Figure 6

Ziram’s toxicity is ZF γ1-dependent. ZF γ1 expression was reduced using a specific morpholino (MO), and a scrambled MO was used as a control. Ziram treatment began at 24 hour post-fertilization (hpf). Neuronal counts (normalized to vehicle + scramble/γ1 MO) for γ1 MO-injected embryos (n = 14). An 86.8% increase in labeled telencephalic VMAT2 neurons (A) and a 45.1% increase in labeled diencepahlic VMAT2 neurons (B) at 3 dpf was observed for fish treated with ziram + γ1 MO versus ziram + scramble MO. *, p < 0.05; **, p < 0.01. Student’s t-test.

Figure 7

CLR01 inhibits ZF γ1 aggregation in vitro and protects against ziram’s toxicity in vivo. Time-dependent change in normalized thioflavin T (ThT) fluorescence over 14 days for 100 μM γ1 incubated in the absence or presence of CLR01 (A). The first and last 130 hr are shown (n = 4). Electron micrographs of ZF γ1 alone (B) and ZF γ1 with CLR01 (C) in an equimolar ratio (scale bar = 100 nm) obtained on day 9 or day 10 of each reaction [analysis of variance (ANOVA), p < 0.0001]. CLR01 treatment protected against ziram toxicity (50 nM; n = 6) to VMAT2 neurons in the diencephalic cluster (E), but the difference did not reach statistical significance in the telencephalic cluster (D) at 5 days post-fertilization (dpf). **, p < 0.01; Student’s t-test.