Synergistic dopaminergic neurotoxicity of the pesticide rotenone and inflammogen lipopolysaccharide: relevance to the etiology of Parkinson's disease

Abstract

Parkinson's disease (PD) is characterized by a progressive degeneration of the nigrostriatal dopaminergic pathway resulting in movement disorders. Although its etiology remains unknown, PD may be the final outcome of interactions among multiple factors, including exposure to environmental toxins and the occurrence of inflammation in the brain. In this study, using primary mesencephalic cultures, we observed that nontoxic or minimally toxic concentrations of the pesticide rotenone (0.5 nm) and the inflammogen lipopolysaccharide (LPS) (0.5 ng/ml) synergistically induced dopaminergic neurodegeneration. The synergistic neurotoxicity of rotenone and LPS was observed when the two agents were applied either simultaneously or in tandem. Mechanistically, microglial NADPH oxidase-mediated generation of reactive oxygen species appeared to be a key contributor to the synergistic dopaminergic neurotoxicity. This conclusion was based on the following observations. First, inhibition of NADPH oxidase or scavenging of free radicals afforded significant neuroprotection. Second, rotenone and LPS synergistically stimulated the NADPH oxidase-mediated release of the superoxide free radical. Third and most importantly, rotenone and LPS failed to induce the synergistic neurotoxicity as well as the production of superoxide in cultures from NADPH oxidase-deficient animals. This is the first demonstration that low concentrations of a pesticide and an inflammogen work in synergy to induce a selective degeneration of dopaminergic neurons. Findings from this study may be highly relevant to the elucidation of the multifactorial etiology of PD and the discovery of effective therapeutic agents for the treatment of the disease.

Fig. 1.

Rotenone and LPS induce synergistic degeneration of dopaminergic neurons. Primary rat neuron–glia cultures were treated with vehicle, indicated concentrations of rotenone or LPS alone, or combinations of indicated concentrations of rotenone and LPS. Eight days later, neurotoxicity was determined by [³H]DA uptake assay and quantification of TH-IR neurons after immunostaining of dopaminergic neurons with an anti-TH antibody. The results are the mean ± SEM of three experiments performed in triplicate.Rot, Rotenone. **p < 0.005 compared with the cultures treated with either rotenone or LPS alone.

Fig. 2.

Rotenone and LPS-induced neurotoxicity is preferential to dopaminergic neurons. Rat neuron–glia cultures were treated with vehicle, 0.5 nm rotenone, and/or 0.5 nm rotenone. Eight days later, cultures were subjected to [³H]DA or [³H]GABA uptake assay (A) or immunostaining with an anti-TH or anti-NeuN antibody followed by quantification of the immunostained neurons (B). The results are the mean ± SEM of three experiments performed in triplicate. R + L, Rotenone plus LPS. **p < 0.005 compared with the control cultures.

Fig. 3.

Double-label immunocytochemical analysis for TH- and NeuN-IR neurons. Rat neuron–glia cultures treated for 8 d with vehicle (Control) or 0.5 nmrotenone and 0.5 ng/ml LPS were double immunostained with anti-TH and anti-NeuN antibodies. The images are from one experiment that is representative of three separate experiments. Scale bar, 50 μm.Arrowheads, TH-IR neurons.

Fig. 4.

Effect of tandem addition of rotenone and LPS on dopaminergic neurodegeneration. A, Tandem addition without change of medium in between. Rat neuron–glia cultures were treated for the indicated periods with rotenone (0.5 nm) and/or LPS (0.5 ng/ml) in the following manner: rotenone or LPS for the entire 8 d (0–8) or only the last 4 d (4–8), rotenone for 8 d (0–8) plus LPS for the last 4 d (4–8), or LPS for 8 d (0–8) plus rotenone for the last 4 d (4–8). Afterward, neurotoxicity was determined by DA uptake and quantification of TH-IR neurons. **p < 0.005 compared with the cultures treated with rotenone alone;⁺p < 0.05 and⁺⁺p < 0.005 compared with the cultures treated with LPS alone. B, Tandem addition with change of medium. Rat neuron–glia cultures were treated for the indicated periods of time with vehicle, 0.5 nm rotenone, and/or 0.5 ng/ml LPS in sequential orders and with medium changes in between as follows: rotenone or LPS alone for the first 3 d only (0–3) or the last 3 d only (5–7), rotenone for the first 3 d (0–3) followed by resting for 1 d and then LPS for 3 d (5–7), LPS for the first 3 d (0–3) followed by resting for 1 d and then rotenone for 3 d (5–7). Afterward, neurotoxicity was determined by DA uptake and quantification of TH-IR neurons. The results are the mean ± SEM of two experiments performed in triplicate. *p < 0.05 compared with the cultures treated with rotenone alone;⁺p < 0.05 compared with the cultures treated with LPS alone. Rot, Rotenone.

Fig. 5.

Induction of synergistic neurotoxicity by rotenone and LPS depends on the presence of glial cells. Rat neuron–glia or neuron-enriched cultures were treated with vehicle, 0.5 nmrotenone, and/or 0.5 ng/ml LPS. Eight days later, neurotoxicity was determined by [³H]DA uptake assay. The results are the mean ± SEM of three experiments performed in triplicate. **p < 0.005 compared with the cultures treated with either rotenone or LPS alone. N/G, Neuron–glia cultures; N/N, neuron-enriched cultures.

Fig. 6.

Role of ROS in the induction of synergistic neurotoxicity by rotenone and LPS. A, Measurement of release of superoxide and levels of intracellular ROS. To measure superoxide release, rat neuron–glia or neuron-enriched cultures were stimulated with vehicle, 0.5 nm rotenone, and/or 0.5 ng/ml LPS. The release of superoxide was measured by the SOD-inhibitable reduction of cytochrome c. The levels of intracellular ROS were detected with CM-H₂-DCFDA. The relative fluorescence intensities in the control cultures were 300–450 and 450–600 arbitrary units per well for N/N andN/G, respectively. The results are expressed as the percentage of control and are the mean ± SEM of two to three experiments performed in triplicate. *p < 0.05 and **p < 0.005 compared with the control cultures, respectively; ⁺⁺p < 0.05 compared with the cultures treated with either rotenone or LPS alone.B, Measurement of the release of superoxide from microglia. Microglia-enriched cultures were pretreated for 30 min with 5 μm DPI or 0.25 mm apocynin before stimulation with vehicle, 0.5 nm rotenone, and/or 0.5 ng/ml LPS. Superoxide released from microglia was determined by measuring the SOD-inhibitable reduction of cytochrome c. The results are the mean ± SEM of three experiments performed in triplicate. *p < 0.05 compared with the vehicle control;⁺p < 0.05 compared with the rotenone and LPS-treated cultures. C, Effect of apocynin,l-NAME, or SOD–catalase on the rotenone and LPS-induced synergistic neurotoxicity. Rat neuron–glia cultures were pretreated for 30 min with vehicle, 0.25 mm apocynin, or 1 mml-NAME before treatment with 0.5 nm rotenone and 0.5 ng/ml LPS. SOD–catalase (100 and 150 U/ml, respectively) were added at the same time with rotenone and LPS. Seven days later, neurotoxicity was determined by [³H]DA uptake assay. The results are the mean ± SEM of three experiments performed in triplicate. **p < 0.005 compared with the vehicle control;⁺p < 0.05 compared with the rotenone and LPS-treated cultures. N/N, Neuron-enriched cultures;N/G, neuron–glia cultures; C, control;R, rotenone; L, LPS; D, DPI; A, apocynin; S/C, SOD–catalase;L-N, l-NAME.

Fig. 7.

Effect of NADPH oxidase deficiency on the induction of neurotoxicity and production of superoxide induced by rotenone and LPS. A, Neurotoxicity. Neuron–glia cultures from wild-type (PHOX^+/+) or NADPH oxidase-deficient (PHOX^−/−) mice were treated with vehicle, 0.5 nm rotenone, and/or 0.5 ng/ml LPS. Eight days later, neurotoxicity was determined by [³H]DA uptake assay. The results are the mean ± SEM of three experiments performed in triplicate. **p < 0.005 compared with the rotenone or LPS-treated cultures. B, Determination of superoxide production in neuron–glia cultures 4 d after treatment. PHOX^+/+ or PHOX^−/−neuron–glia cultures were treated with vehicle, 0.5 nmrotenone, and/or 0.5 ng/ml LPS. Four days later, levels of superoxide were measured as SOD-inhibitable cytochrome c reduction. **p < 0.005 compared with the vehicle control;⁺⁺p < 0.005 compared with the cultures treated with either rotenone or LPS alone. C, Production of superoxide in microglia. PHOX^+/+ or PHOX^−/− microglia-enriched cultures were stimulated with vehicle, 0.5 nm rotenone, and/or 0.5 ng/ml LPS. Release of superoxide were measured as SOD-inhibitable cytochromec reduction. **p < 0.005 compared with the vehicle control; ⁺⁺p < 0.005 compared with the cultures treated with either rotenone or LPS alone.C, Control; R, rotenone;L, LPS.