Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease

Abstract

Loss-of-function mutations of the parkin gene are a major cause of early-onset parkinsonism. To explore the mechanism by which loss of parkin function results in neurodegeneration, we are using a genetic approach in Drosophila. Here, we show that Drosophila parkin mutants display degeneration of a subset of dopaminergic (DA) neurons in the brain. The neurodegenerative phenotype of parkin mutants is enhanced by loss-of-function mutations of the glutathione S-transferase S1 (GstS1) gene, which were identified in an unbiased genetic screen for genes that modify parkin phenotypes. Furthermore, overexpression of GstS1 in DA neurons suppresses neurodegeneration in parkin mutants. Given the previous evidence for altered glutathione metabolism and oxidative stress in sporadic Parkinson's disease (PD), these data suggest that the mechanism of DA neuron loss in Drosophila parkin mutants is similar to the mechanisms underlying sporadic PD. Moreover, these findings identify a potential therapeutic approach in treating PD.

Fig. 1.

A subset of DA neurons degenerates in parkin mutants. (A) Diagram shows the locations of the major DA neuron clusters in the adult Drosophila brain. PAL, protocerebral anterior lateral; PPM, protocerebral posterior medial; VUM, ventral unpaired medial. The PAL cluster is located in the same position but anterior to the PPL1 cluster, as designated by the arrow. (B) A projected Z-series confocal image from a WT adult brain stained with anti-TH to label DA neurons. DA neurons within the boxed section are shown at higher magnification in C and D. (C and D) Representative images from WT (C) and parkin mutant (D) brains aged 20 days. Fewer DA neurons are detected in the PPL1 cluster in parkin mutants relative to WT. (E and F) The number of DA neurons in each major DA neuron cluster in WT (black bars) and parkin mutants (gray bars) in 1-day-old (E) and 20-day-old (F) adult flies. parkin mutants display a significant decrease in the number of PPL1 neurons at 1 day of age (*, P < 0.05). Neuron loss in parkin mutants is more extensive in the PPL1 cluster at 20 days of age (***, P < 0.0001). Statistical significance was calculated by using Student's t test. n refers to the number of brains used for neuron counts. Animals homozygous for the parkin null allele park²⁵ or bearing the isogenic park^rvA chromosome, which has a WT allele of parkin, were used in these analyses.

Fig. 2.

Neuron loss in parkin mutants is specific to DA neurons and results primarily from loss of a cell-autonomous requirement of parkin.(A–C) Expression of GFP driven by TH-GAL4 (A) and anti-TH (B) staining reveals a high degree of colocalization of signals (C). The boxed area in C marks the PPL1 cluster and is magnified in C′.(D) Analysis of GFP-positive neurons in 20-day-old adult parkin mutants and age-matched WT controls bearing the TH-GAL4 driver and a UAS-GFP transgene confirms the cell loss in the PPL1 DA neuron cluster of parkin mutants (**, P < 0.005, Student's t test). (E) DA neuron loss is significantly reduced by expression of a parkin transgene using the TH-GAL4 driver compared with parkin mutant or parkin mutants bearing the TH-GAL4 driver alone. Expression of GAL4 from the TH-GAL4 driver did not significantly affect DA neuron viability. (**, P < 0.001; Δ, P = 0.3) (F) Diagram shows all major serotonergic neurons in the adult brain. SP, subaesophageal; LP, lateral protocerebral; IP, inferior medial protocerebral. (G) Representative confocal micrograph of a WT adult brain stained with anti-5-hydroxytyrosine to reveal serotonergic neurons. (H) No significant difference in the number of serotonergic neurons is detectable in 20-day-old adult parkin mutants relative to age-matched controls, indicating that neuron loss is specific to a subset of DA neurons. Statistical significance was calculated by using ANOVA and Bonferroni's post hoc test for planned comparisons. Numbers shown in histograms refer to the number of brains used for neuron counts. park²⁵ or park^rvA homozygotes were used in all analyses of neuronal viability.

Fig. 3.

GstS1 activity modifies locomotor deficits in parkin mutants. (A) Loss-of-function alleles of GstS1 enhance the climbing defect of parkin mutants. Each loss-of-function GstS1 allele, EP2223, k09303, k08805, and 04227, used in this analysis was heterozygous with a WT allele of GstS1. park²⁵ or park^rvA homozygotes were used in all climbing assays. Enhancement of the parkin climbing defect by each of the GstS1 alleles was significant (P < 0.005, Student's t test). (B) Directed expression of a GstS1 transgene (UAS-GstS1) by a muscle GAL4 transgene (24B) alleviates the partial climbing deficits seen in two parkin hypomorphic mutants (park^Z472 and park^Z3678). Suppression of climbing deficits from GstS1 overexpression was significant (**, P < 0.001) by Student's t test.

Fig. 4.

GstS1 activity influences DA neuron viability in parkin mutants. (A)A GstS1 null allele (GstS1^M26) in trans to a deletion (Df) of the GstS1 region [Df(2R)ED1] enhances DA neuron loss detected in the PPL1 cluster of 1-day-old parkin mutants. GstS1 null mutants (GstS1^M26/Df) alone manifest no DA neuron loss in a WT parkin background. (B) Transgenic overexpression of GstS1 in DA neurons significantly suppresses DA neuron loss in 20-day-old parkin mutants (park²⁵,TH-G4; UAS-GstS1). The degree of rescue conferred by GstS1 is comparable to that achieved by transgenic expression of parkin in DA neurons (park²⁵,TH-G4; UAS-park). Statistical significance was calculated by using ANOVA and Bonferroni's post hoc test for planned comparisons. (*, P <0.05; **, P < 0.001; Δ, P = 0.3). Numbers shown in bars refer to the number of brains analyzed for neuron counts. park²⁵ or park^rvA homozygotes were used in all analyses of neuronal viability.