Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin

Abstract

Mutations in Pink1, a gene encoding a Ser/Thr kinase with a mitochondrial-targeting signal, are associated with Parkinson's disease (PD), the most common movement disorder characterized by selective loss of dopaminergic neurons. The mechanism by which loss of Pink1 leads to neurodegeneration is not understood. Here we show that inhibition of Drosophila Pink1 (dPink1) function results in energy depletion, shortened lifespan, and degeneration of select indirect flight muscles and dopaminergic neurons. The muscle pathology was preceded by mitochondrial enlargement and disintegration. These phenotypes could be rescued by the wild type but not the pathogenic C-terminal deleted form of human Pink1 (hPink1). The muscle and dopaminergic phenotypes associated with dPink1 inactivation show similarity to that seen in parkin mutant flies and could be suppressed by the overexpression of Parkin but not DJ-1. Consistent with the genetic rescue results, we find that, in dPink1 RNA interference (RNAi) animals, the level of Parkin protein is significantly reduced. Together, these results implicate Pink1 and Parkin in a common pathway that regulates mitochondrial physiology and cell survival in Drosophila.

Fig. 1.

Inhibition of dPink1 by RNAi causes defects of abnormal wing posture, shortened lifespan, and ATP deficit. (A and B) RT-PCR and Western blot analyses of dPink1 mRNA and protein levels after RNAi. Genotypes: 1, Da-Gal4/+; 2, Da-Gal4>dPink1 RNAi. RP49 and tubulin serve as controls. (C–E) Wing posture phenotypes in control and dPink1 RNAi flies. Left, females; right, males. Flies were 7 days old and kept at 29°C. (C) Straight wing posture of control flies. (D) dPink1 RNAi flies displaying held-up wings. (E) dPink1 RNAi flies displaying drooped wings. (F) Abolished flight activities in dPink1 RNAi flies. Two independent lines were used. (G) dPink1 RNAi flies exhibit a sharper age-dependent decline of ATP content. (H) Female dPink1 flies exhibit a shortened lifespan. (A–H) RNAi was achieved by using a UAS-dPink1 RNAi construct driven by Da-Gal4. ∗, P < 0.01 in Student’s t test.

Fig. 2.

Inhibition of dPink1 results in disrupted IFMs. (A-H) Light microscopy was used to examine IFM architecture (stars, dorsal longitudinal muscles; arrows, dorsal ventral muscles). Sections from resin-embedded thoraces of 1-week-old adult flies were stained with toluidine blue to visualize tissue morphology; anterior is to the left. (I–N) Wing posture phenotypes of control and dPink1 RNAi flies directed by Mhc-Gal4. Fly genotypes are Da-Gal4/+ (A), Da-Gal4>UAS-dPink1 RNAi (B), Mhc-Gal4/+ (C and I), Mhc-Gal4>UAS-dPink1 RNAi (D and J), Mhc-Gal4>UAS-dPink1 RNAi; UAS-dPink1 (E and K), Mhc-Gal4>UAS-dPink1 RNAi; UAS-GFP (F and L), Mhc-Gal4>UAS-dPink1 RNAi; UAS-hPink1 (G and M), Mhc-Gal4>UAS-dPink1 RNAi; UAS-hPink1ΔC (H and N).

Fig. 3.

Muscle-specific dPink1 RNAi results in myopathology and age-dependent apoptosis in the IFMs. (A–D) EM analysis of IFM ultrastructure of flies with the following genotypes: Mhc-Gal4/+ (A), Mhc-Gal4>UAS-dPink1 RNAi (B), Mhc-Gal4>UAS-dPink1 RNAi; UAS-hPink1 (C), and Mhc-Gal4>UAS-dPink1 RNAi; UAS- hPink1ΔC (D). Arrows, swollen mitochondria; arrowheads, rod body-like deposits. (E and F) EM analysis of TTM ultrastructure in Mhc-Gal4/+ (E) and Mhc-Gal4>UAS-dPink1 RNAi (F) flies. Scale bars (5 μm) are shown at the lower left corner of each image. (G and H) TUNEL staining of thoracic musculatures from newly eclosed (G) and 1-week-old (H) Mhc-gal4>dPink1 RNAi flies. Left, DAPI staining; Center, TUNEL staining; Right, merged images. Arrows point to TUNEL-positive nuclei.

Fig. 4.

Dopaminergic defects in dPink1 RNAi flies. (A–E) Whole-mount brain TH immunostaining of dorsolateral protocerebral posterior (PPL1) cluster neurons in 25-day-old flies of the following genotypes: TH-Gal4/+ (A), TH-Gal4/UAS-dPink1 RNAi; UAS-GFP (B), TH-Gal4/UAS-dPink1 RNAi; UAS- hPink1ΔC (C), TH-Gal4/UAS-dPink1 RNAi; UAS-hPink1 (D), and TH-Gal4/UAS-dPink1 RNAi; UAS-hParkin (E). (F) Quantification of TH+ neurons in individual dopaminergic clusters in the adult brains of the flies with the indicated genotypes. ∗, P < 0.01 in Student’s t test. (G) Quantification of head dopamine levels in TH-Gal4/+, TH-Gal4/UAS-White RNAi, or TH-Gal4/UAS-dPink1RNAi flies, with white RNAi flies serving as control. ∗, P < 0.01 in Student’s t test.

Fig. 5.

Genetic and biochemical interaction between Pink1 and Parkin. Wing posture (A and B), thorax musculature histology (C and D), and IFM EM images (E and F) of Mhc-Gal4/UAS-dPink1 RNAi; UAS-hParkin (A, C, and E), and Mhc-Gal4/UAS-dPink1 RNAi; UAS-hDJ-1 (B, D, and F) flies are shown. Scale bars (2 μm) in E and F are shown at the bottom left corner. (G) Whole-body ATP measurements of the indicated genotypes. ∗, P < 0.01 in Student’s t test. (H) Western blot analysis showing reduction of dParkin levels in dPink1 RNAi animals. Protein extracts prepared from Da-Gal4/+ and Da-Gal4/UAS-dPink1 RNAi animals were probed with anti-dParkin antibody. Actin serves as protein loading control.