Distinct role for microglia in rotenone-induced degeneration of dopaminergic neurons

Abstract

Increasing evidence has suggested an important role for environmental factors such as exposure to pesticides in the pathogenesis of Parkinson's disease. In experimental animals the exposure to a common herbicide, rotenone, induces features of parkinsonism; mechanistically, rotenone-induced destruction of dopaminergic neurons has been attributed to its inhibition of the activity of neuronal mitochondrial complex I. However, the role of microglia, the resident brain immune cells in rotenone-induced neurodegeneration, has not been reported. Using primary neuron-enriched and neuron/glia cultures from the rat mesencephalon, we discovered an extraordinary feature for rotenone-induced degeneration of cultured dopaminergic neurons. Although little neurotoxicity was detected in neuron-enriched cultures after treatment for 8 d with up to 20 nm rotenone, significant and selective dopaminergic neurodegeneration was observed in neuron/glia cultures 2 d after treatment with 20 nm rotenone or 8 d after treatment with 1 nm rotenone. The greatly enhanced neurodegenerative ability of rotenone was attributed to the presence of glia, especially microglia, because the addition of microglia to neuron-enriched cultures markedly increased their susceptibility to rotenone. Mechanistically, rotenone stimulated the release of superoxide from microglia that was attenuated by inhibitors of NADPH oxidase. Furthermore, inhibition of NADPH oxidase or scavenging of superoxide significantly reduced the rotenone-induced neurotoxicity. This is the first report demonstrating that microglia play a pivotal role in rotenone-induced degeneration of dopaminergic neurons. The results of this study should advance our understanding of the mechanism of action for pesticides in the pathogenesis of Parkinson's disease.

Fig. 1.

Differential sensitivity of dopaminergic neurons in neuron-enriched and mixed neuron/glia mesencephalic cultures to rotenone-induced degeneration. A, B, Primary neuron-enriched or neuron/glia cultures were treated for 8 d with the vehicle (control group) or 1–30 nm rotenone; degeneration of dopaminergic neurons was evaluated with the [³H]DA uptake assay (A) or quantification of TH-IR neurons in the cultures (B). C, Mixed neuron/glia cultures were treated for 2–8 d with the vehicle or 1–25 nmrotenone; the [³H]DA uptake was determined at the desired time points. The amount of DA uptake for the vehicle-treated neuron/glia cultures at the time of treatment and 8 d after treatment was 0.77 ± 0.04 and 0.65 ± 0.06 and for vehicle-treated neuron-enriched cultures was 0.81 ± 0.07 and 0.77 ± 0.08 pmol/min per well, respectively. The results for DA uptake (A, C) and cell counts (B) are expressed as a percentage of the control cultures and are the mean ± SEM of four experiments performed in triplicate. *p < 0.01, compared with the control.

Fig. 2.

Selectivity of rotenone-induced neurodegeneration in mixed neuron/glia cultures. A, B, Mixed neuron/glia cultures were treated for 8 d with the vehicle or 5–25 nm rotenone. Afterward the cultures were assayed for uptake of [³H]DA or [³H]GABA (A) or were immunostained with anti-TH, Neu-N, or MAP-2 antibodies, followed by quantification of the positively stained cells (B). The amount of GABA uptake at the time of treatment and 8 d after treatment for the vehicle-treated neuron/glia cultures was 39.4 ± 4.2 and 35.2 ± 3.2 pmol/min per well, respectively. The number of Neu-N-IR, MAP-2-IR, and TH-IR neurons in the control cultures was ∼490, 460, and 14/mm². The results are expressed as a percentage of the control cultures and are the mean ± SEM of three experiments performed in triplicate. *p < 0.01, compared with the control. C, Immunocytochemical analysis. Mixed neuron/glia cultures were treated for 8 d with the vehicle or 10 or 20 nm rotenone. Cultures then were immunostained for TH-IR, Neu-N-IR, or MAP-2-IR neurons or were double stained for TH-IR and Neu-N-IR neurons. Scale bars: TH-IR and Neu-N-IR neurons, 50 μm; MAP-IR neurons, 100 μm; neurons in the TH and Neu-N double-immunostained cultures, 75 μm.

Fig. 3.

Effect of the addition of microglia to neuron-enriched cultures on rotenone-induced degeneration of dopaminergic neurons. Neuron-enriched mesencephalic cultures were supplemented with 2.5–7.5 × 10⁴microglia/well. At 24 hr later the cultures were treated with the vehicle or 10 nm rotenone, and DA uptake was determined 8 d after the treatment. The results are expressed as a percentage of the control cultures and are the mean ± SEM of three experiments performed in triplicate. *p < 0.01, compared with the control.

Fig. 4.

Effect of rotenone on microglial activation. Mixed neuron/glia cultures were treated for 1 d with the vehicle or 1–10 nm rotenone and then were immunostained with antibodies against specific markers of microglia. A, Immunocytochemical analysis with the OX-42 antibody of cultures treated for 1 d with the vehicle or 10 nm rotenone. In control cultures a significant portion of the OX-IR resting microglia was small and round (open arrowheads). After treatment with 10 nm rotenone, OX-42-IR microglia were enlarged significantly and were irregularly shaped (filled arrowheads), indicative of activation. Part of the images of OX-42-IR microglia (inclosed boxes, a and b) presented in A was cut out and presented inB for better representation of the differences.C, Quantification of microglial activation. Vehicle or rotenone-treated cultures were immunostained with OX-42, ED-1, or isolectin; positively stained microglia were counted. The number of OX-42-IR microglia in the control cultures was ∼120/mm². The results are expressed as a percentage of the control cultures and are the mean ± SEM of three experiments performed in triplicate. *p< 0.01, compared with the control. Scale bar, 50 μm.

Fig. 5.

Rotenone stimulated the release of superoxide.A, Rat primary microglia, human monocytic U937 cells, or rat neutrophils were seeded to 96-well plates. Cells were treated with the vehicle or 1–10 nm rotenone. Superoxide production, measured as SOD-inhibitable cytochrome c reduction, was determined as described in Materials and Methods. The results are a percentage of the control cultures and are expressed as the mean ± SEM of three experiments performed in triplicate. *p < 0.01, compared with the control.B, Effect of NADPH oxidase inhibitors on rotenone-induced release of superoxide from neutrophils. Neutrophils were pretreated for 5 min with the vehicle or indicated concentrations of DPI or apocynin before treatment with 10 nm rotenone. Then the amount of superoxide released was determined as described in Materials and Methods. The results are a percentage of the control cultures and are expressed as the mean ± SEM of three experiments performed in triplicate. *p < 0.01, compared with rotenone-treated cells. C, Control; Rot, rotenone; APO, apocynin.

Fig. 6.

Effect of SOD/catalase on rotenone-induced degeneration of dopaminergic neurons. SOD/catalase (50 U/ml each) were added to neuron/glia cultures right before and 24 hr after the addition of the indicated concentrations of rotenone. The cultures were assayed for [³H]DA uptake 8 d later. The results are the mean ± SEM of three experiments performed in triplicate. *p < 0.01, compared with the control.

Fig. 7.

Effect of apocynin on rotenone-induced degeneration of dopaminergic neurons. A, B, Neuron/glia cultures were pretreated for 30 min with the vehicle or 0.25 mm apocynin before treatment with 5 or 10 nmrotenone. Then 8 d later the degeneration of dopaminergic neurons was assessed as [³H]DA uptake (A) and counts of TH-IR neurons (B). C, Neuron/glia cultures were pretreated for 30 min with the vehicle or the indicated concentration of apocynin before treatment with 10 nm rotenone. The cultures were assayed for [³H]DA uptake 8 d later. The results are the mean ± SEM of three experiments performed in triplicate. *p < 0.01 and⁺p < 0.05, compared with the control.