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. 2006 Sep 5;103(36):13520-5.
doi: 10.1073/pnas.0604661103. Epub 2006 Aug 24.

Antioxidants protect PINK1-dependent dopaminergic neurons in Drosophila

Danling Wang  1 , Li Qian, Hui Xiong, Jiandong Liu, Wendi S Neckameyer, Sean Oldham, Kun Xia, Jianzhi Wang, Rolf Bodmer, Zhuohua Zhang
Affiliations

Antioxidants protect PINK1-dependent dopaminergic neurons in Drosophila

Danling Wang et al. Proc Natl Acad Sci U S A. .

Abstract

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder. Mutations in the PINK1 gene are linked to the autosomal recessive early onset familial form of PD. The physiological function of PINK1 and pathological abnormality of PD-associated PINK1 mutants are largely unknown. We here show that inactivation of Drosophila PINK1 (dPINK1) using RNAi results in progressive loss of dopaminergic neurons and in ommatidial degeneration of the compound eye, which is rescued by expression of human PINK1 (hPINK1). Expression of human SOD1 suppresses neurodegeneration induced by dPINK1 inactivation. Moreover, treatment of dPINK1 RNAi flies with the antioxidants SOD and vitamin E significantly inhibits ommatidial degeneration. Thus, dPINK1 plays an essential role in maintaining neuronal survival by preventing neurons from undergoing oxidative stress, thereby suggesting a potential mechanism by which a reduction in PINK1 function leads to PD-associated neurodegeneration.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Knockdown of dPINK1 results in age-dependent loss of DA neurons, but not serotonergic neurons, in Drosophila. Brains dissected from flies expressing elav-GAL4 driver alone (A, E, and L), elav-GAL4/UAS-lac Z (B, F, and M), elav-GAL4/UAS-hPINK1 (C, G, and N), elav-GAL4/UAS-dPINK1 RNAi (D, H, and O), and elav-GAL4/UAS-dPINK1 RNAi/UAS-hPINK1 (K) aged 1 day (A–D) or 10 days (E–G, K, and L–O) were immunostained with either anti-Drosophila TH antibody or anti-5HT antibody followed by an Alexa Fluor 594-labeled secondary antibody to identify DA (A–H and K) or serotonergic (L–O) neurons, respectively. Representative pictures shown were collected by using confocal microscopy. Localization of Drosophila DA and serotonergic neurons are illustrated in I and J, respectively. Note that changes of DA neurons in VUM (white circle) and PPL2 (yellow circle) regions in 1-day (D) and 10-day (H) fly brains with dPINK1 knockdown as well as 10-day fly brains (K) with dPINK1 knockdown rescued by hPINK1 are indicated. Serotonergic neurons remain similar among all groups (L–O). (Scale bar, 50 μm.)
Fig. 2.
Fig. 2.
Knockdown of dPINK1 induces degeneration of ommatidia and retinal neurons in Drosophila. External eye phenotypes of flies aged 1 day expressing GMR-GAL4 driver alone (A, A′, and A″), GMR-GAL4/UAS-lac Z (B, B′, and B″), GMR-GAL4/UAS-dPINK1 RNAi (C, C′, C″), GMR-GAL4/UAS-hPINK1 (D and D′), GMR-GAL4/UAS-hPINK1G309D (D″), GMR-GAL4/UAS-dPINK1 RNAi/UAS-hPINK1 (E and E′), and GMR-GAL4/UAS-dPINK1 RNAi/UAS-hPINK1 G309D (E″) under light microscopy (A–E and A″–E″) and electronic microscopy (A′–E′). Magnification of light microscopic images and electronic microscopic images is ×25 and ×400, respectively. Arrows indicate lesions of ommatidial degeneration (C, C′, C″, and E″). Microscopic images of DLG staining (F–I) as well as phalloidin and elav double staining of 44 h AFP retina dissected from flies expressing GMR-GAL4 driver alone (F and J), GMR-GAL4/UAS-lac Z (G and K), and GMR-GAL4/UAS-dPINK1 RNAi (H, I, L, and M). Images from two independent lines expressing GMR-GAL4/UAS-dPINK1 RNAi are shown (H and L are from one line, whereas I and M are from another independent line). Note that dPINK1 knockdown induces cellular disorganization (H and I) and loss of neurons (L and M; yellow arrows). (Scale bar, 10 μm.)
Fig. 3.
Fig. 3.
Expression of hSOD1 suppresses degeneration of ommatidia and DA neurons. Representative images of external eye phenotypes of 3-day-old flies expressing GMR-GAL4 driver alone (A), GMR-GAL4/UAS-lac Z (B), GMR-GAL4/UAS-dPINK1 RNAi (C), and GMR-GAL4/UAS-dPINK1 RNAi/UAS-hSOD1 (D), as well as images of DA neurons identified by anti-Drosophila TH staining of 10-day-old flies expressing elav-GAL4 driver alone (A′), elav-GAL4/UAS-lac Z (B′), elav-GAL4/UAS-dPINK1 RNAi (C′), and elav-GAL4/UAS-dPINK1 RNAi/UAS-hSOD1 (D′). Note that ommatidial degeneration (C, yellow arrow) and loss (C′) and rescue (D′) of DA neurons in PPM1/2 (white circle) and PPL2 (yellow circle) are indicated. (Scale bar, 50 μm.) (E) Quantification of DA neurons is shown for 10-day-old flies expressing elav-GAL4 driver alone (WT), elav-GAL4/UAS-lac Z (lac Z), elav-GAL4/UAS-hPINK1 (hPINK1), elav-GAL4/UAS-hSOD1 (SOD1), elav-GAL4/UAS-dPINK1 RNAi (dPINK1KD), elav-GAL4/UAS-dPINK1 RNAi/UAS-lac Z (dPINK1KD, lacZ), elav-GAL4/UAS-dPINK1 RNAi/UAS-hPINK1 (dPINK1KD, hPINK1), and elav-GAL4/UAS-dPINK1 RNAi/UAS-hSOD1 (dPINK1KD, SOD1). DA neurons in six brain regions, including PAL, PPM1/2, PPM3, PPL1, PPL2, and VUM, were quantitated, and differences were statistically analyzed. ∗, P < 0.05. ns, no statistical significance.
Fig. 4.
Fig. 4.
Inhibition of ommatidial degeneration by the antioxidants SOD1 and vitamin E. Flies (7 days old) with dPINK1 knockdown driven by GMR-GAL4 (GMR-GAL4/UAS-dPINK1 RNAi) were treated without (A) or with 100 units/ml (B) and 1,000 units/ml (C) SOD, or without (A′) or with 20 μg/ml (B′) and 200 μg/ml (C′) vitamin E. Representative images of external eye phenotype of each group are shown. (Magnification, ×25.) Flies overexpressing hPINK1 were treated with either 20 mM paraquat for 24 h (D) or 1% H2O2 over 6 days (E) and were quantitated for survival rates. Results from lines expressing Da-GAL4 driver alone (Da-GAL4) or Da-GAL4/UAS-lac Z (lac Z) and two independent lines expressing Da-GAL4/UAS-hPINK1 (hPINK1FC2 and hPINK1TC1) are shown. ∗∗, P < 0.001.
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