A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila

Abstract

Long-term exposure to environmental oxidative stressors, like the herbicide paraquat (PQ), has been linked to the development of Parkinson's disease (PD), the most frequent neurodegenerative movement disorder. Paraquat is thus frequently used in the fruit fly Drosophila melanogaster and other animal models to study PD and the degeneration of dopaminergic neurons (DNs) that characterizes this disease. Here, we show that a D1-like dopamine (DA) receptor, DAMB, actively contributes to the fast central nervous system (CNS) failure induced by PQ in the fly. First, we found that a long-term increase in neuronal DA synthesis reduced DAMB expression and protected against PQ neurotoxicity. Secondly, a striking age-related decrease in PQ resistance in young adult flies correlated with an augmentation of DAMB expression. This aging-associated increase in oxidative stress vulnerability was not observed in a DAMB-deficient mutant. Thirdly, targeted inactivation of this receptor in glutamatergic neurons (GNs) markedly enhanced the survival of Drosophila exposed to either PQ or neurotoxic levels of DA, whereas, conversely, DAMB overexpression in these cells made the flies more vulnerable to both compounds. Fourthly, a mutation in the Drosophila ryanodine receptor (RyR), which inhibits activity-induced increase in cytosolic Ca(2+), also strongly enhanced PQ resistance. Finally, we found that DAMB overexpression in specific neuronal populations arrested development of the fly and that in vivo stimulation of either DNs or GNs increased PQ susceptibility. This suggests a model for DA receptor-mediated potentiation of PQ-induced neurotoxicity. Further studies of DAMB signaling in Drosophila could have implications for better understanding DA-related neurodegenerative disorders in humans.

Figure 1.

Paraquat-induced lethality and morphological alterations of Drosophila DA neurons. (A and B) Survival rate of wild-type Canton-S Drosophila either fed with a sucrose solution containing PQ (dietary ingestion) (A) or after a 5-s application of the drug diluted in Ringer's solution to the VNC of decapitated flies (direct application) (B). PQ concentrations were as follows: 0 (closed circles), 20 (open squares), or 80 mm (closed squares). (C) Structure of brain DNs from wild-type Drosophila fed for 24 h on a sucrose solution containing either no PQ (control, upper panel) or 20 mm PQ (lower panel). Brains were then dissected and stained with anti-TH antibodies. The pictures show part of the dorsolateral posterior protocerebral 2 (PPL2) neuronal cluster. Dopaminergic cell bodies of PQ-treated flies appeared smaller and rounder with shrunken nuclei. (D and E) Whole-mount VNC of decapitated Drosophila dissected 2 h after a 5-s application of either Ringer's alone (control) (D) or 80 mm PQ (E) and then immunostained for TH. Confocal projections were converted into negative images to improve visibility. DN axonal varicosities that are widespread in the VNC of control flies appear to be much reduced in size or lost (98.4 ± 0.005%) in PQ-treated VNC. Scale bars: (C) 10 µm, (D and E) 50 µm.

Figure 2.

Down-regulation of the DA receptor DAMB protects Drosophila against PQ-induced oxidative stress. (A) Survival rate of wild-type flies fed for 24 h with sucrose plus 20 mm PQ, compared with TH-GAL4; UAS-DTHg flies overexpressing TH in DNs (TH>THg), and TRH-GAL4; UAS-DTHg flies ectopically expressing TH in serotonergic neurons (TRH>THg). (B) Survival rate of decapitated flies of similar genotypes as in (A) 2 h after a 5-s application of 80 mm PQ dissolved in Ringer's to the VNC. (C) Effect of DA receptor antagonists. Survival rate of decapitated flies was monitored 1 h 30 min after brief application of a drop of Ringer's solution containing either PQ alone (control) or PQ plus D₁ antagonists SKF-83959 or SKF-83566, or D₂ antagonist eticlopride. Protection was observed with the D₁-specific antagonists. (D) PQ susceptibility of decapitated D₁-like DA receptor mutants. Whereas the survival rate of dumb² is comparable with wild type, the Damb¹ strain shows higher resistance to PQ. (E) Survival of intact Damb¹ flies after PQ ingestion is similarly increased compared with wild type. In (A)–(E), values were normalized to the mean survival rate of wild-type controls. (F–H) TH overexpression decreases the DAMB transcript level in adult flies. (F) RT-PCR from head RNAs (30 cycles of amplification) showed reduced level of DAMB transcripts, compared with wild type, after long-term TH overexpression in dopaminergic (TH>THg) or serotonergic (TRH>THg) neurons, and their absence in the Damb¹ mutant. Density analysis of RT-PCR products (G) or real-time PCR experiments (H) yielded similar quantitative results.

Figure 3.

Age-related increase in Drosophila PQ susceptibility appears to be DAMB dependent. (A) Whereas newly eclosed (1-day-old) wild-type (w¹¹¹⁸) Drosophila were quite resistant to PQ exposure, susceptibility markedly increased during the first 2 weeks of adult life. In contrast, resistance of w; Damb¹ mutant flies after PQ ingestion was similarly high at all ages during this period. (B) RT-PCR experiments (30 cycles of amplification) demonstrated that DAMB expression progressively increased with age in wild-type flies during the first 2 weeks after adult eclosion. (C) Quantification of the RT-PCR products from 3 to 5 independent experiments by density analysis. The relative transcript abundance of the DA receptor in 15-day-old flies was found to be about twice as much as in 1-day-old flies.

Figure 4.

DAMB is expressed in GNs of the VNC. (A) Sketch of Glu- (green circles) and DA- (red circles) synthesizing cells in the VNC. The regions corresponding to the thoracic ganglia (tho) and fused abdominal neuromeres (abd) are marked. Circles represent neuronal cell bodies. The gray rectangle indicates the region magnified in panels (C) and (D), and the dashed rectangle does the same for panels (E) and (G). (B) RT-PCR experiment (40 cycles) showing that DAMB is expressed both in the head and thorax of wild-type Canton-S flies and is absent from both tissues in Damb¹ mutant. (C) Presence of DAMB immunoreactivity in adult VNC of wild-type flies. DAMB is expressed particularly in neuronal cell bodies that may correspond to Glu-releasing motor neurons. (D) No DAMB immunoreactivity was detected in VNC of Damb¹ mutant. (E–G) Double staining with anti-DAMB (magenta) and anti-GFP (green) antibodies in VNC of VGlut-GAL4; UAS-mCD8::GFP flies, (E) anti-DAMB, (F) merge and (G) anti-GFP. Large arrows show cell bodies of GNs expressing DAMB at various levels. Thin arrows indicate cells expressing DAMB and not GFP. (H–J) Double staining with anti-TH (magenta) and anti-GFP (green) antibodies in VNC of VGlut-GAL4; UAS-VGlut::GFP flies. (H) anti-TH, (I) merge and (J) anti-GFP. DN axonal varicosities and GN nerve endings are widely distributed and overlap in several regions of the VNC neuropil. (K–M) Magnification of the regions indicated by white rectangles in I. Examples of overlapping neuronal domains are marked (arrowheads). Scale bars: (C and D) 25 µm, (E–G) 5 µm, (H–J) 50 µm and (K–M) 20 µm.

Figure 5.

DAMB inactivation in GNs enhances PQ tolerance. (A and B) Survival of decapitated (A) and intact (B) flies at 2 and 48 h, respectively, after PQ exposure. The survival rate is greater for VGlut-GAL4; UAS-DAMB-IR Drosophila (VGlut>DAMB-IR), which express DAMB interfering RNA (IR) in GNs compared with control flies carrying one copy of VGlut-GAL4 or UAS-DAMB-IR alone. (C) Survival of decapitated TH-GAL4; UAS-DAMB-IR flies (TH>DAMB-IR) that express DAMB IR in DNs is, in contrast, similar to controls. (D) PQ susceptibility of VGlut-GAL4; UAS-DAMB flies (VGlut>DAMB) that overexpress DAMB in GNs is significantly increased compared with controls. (E and F) Effect of in vivo stimulation of Glu- or DA-releasing neurons on PQ resistance. (E) Survival of decapitated VGlut-GAL4; UAS-dTrpA1 (VGlut>dTrpA1) Drosophila incubated for 2 h at 31°C in the presence of PQ is decreased compared with similarly treated control flies carrying one copy of either VGlut-GAL4 or UAS-dTrpA1 alone. (F) Same experiment as in D with TH-GAL4; UAS-dTrpA1 flies (TH>dTrpA1). Short-term stimulation of DNs also markedly increased PQ neurotoxicity. In all panels, values were normalized to the mean survival rate of respective UAS strain controls.

Figure 6.

DAMB inactivation protects decapitated Drosophila against DA neurotoxicity. (A) Survival of decapitated wild-type (Canton-S) flies was monitored at various times (15, 30, 45 and 60 min) after a 5-s application of a drop of DA dissolved in Ringer's at the indicated concentration (10, 20, 50 or 100 mm). DA levels of >10 mm were found to be toxic in a dose-dependent manner under these conditions. (B) Survival rate of decapitated flies 2 h after a 5-s application of Ringer's solution alone (R) or 35 mm DA dissolved in Ringer's (DA). Damb¹ mutants resist DA toxicity better than wild-type flies. (C) Survival of decapitated Drosophila 2 h after a 5-s exposure to 35 mm DA is significantly prolonged for VGlut-GAL4; UAS-DAMB-IR flies (VGlut>DAMB-IR) that express DAMB interfering RNA in GNs as compared with control flies carrying one copy of VGlut-GAL4 or UAS-DAMB-IR alone. (D) DA susceptibility of VGlut-GAL4; UAS-DAMB Drosophila (VGlut>DAMB) that overexpress DAMB in Glu neurons is markedly increased compared with controls. Values were normalized to the mean survival rate of UAS strain controls. Note that all experiments in this figure were performed in the absence of PQ.

Figure 7.

Mechanisms of DAMB-mediated potentiation of PQ susceptibility in Drosophila. (A) Survival of decapitated wild-type Drosophila was monitored at various times after a 5-s application of a PQ solution mixed (open squares) or not (closed squares) with forskolin plus IBMX, as described in Materials and Methods. Increased cAMP levels appeared to protect the flies against PQ neurotoxicity. (B) Heterozygous RyR¹⁶ mutants that have a reduced level of ryanodine receptor calcium channels survived in significantly higher numbers compared with wild-type flies either at 24 or 48 h after PQ ingestion. This suggests a primary role for cytosolic Ca²⁺ in the PQ toxicity pathway. (C) Proposed model of PQ neurotoxicity in flies. (a) In physiological conditions, DA release and DAMB-mediated signaling are not neurotoxic. (b) In naïve flies exposed to PQ, high amounts of DA released from oxidative stress-injured DNs would overactivate DAMB. Neuronal dysfunction would then result from two synergistic causes: on the one hand, elevated oxidative stress caused by PQ absorption, and on the other hand, aberrant DAMB activation leading to increased cytosolic Ca²⁺ through the ryanodine receptor. This would ultimately cause CNS failure and precipitate organism death. (c) In Damb¹ mutants or in flies with constitutively higher DA synthesis, DAMB deficiency or DAMB down-regulation, respectively, alleviate PQ neurotoxicity, mediating CNS protection and prolonged Drosophila survival. Such a modulation of DAMB expression might be used in wild-type flies as an adaptive mechanism to increase oxidative stress tolerance.