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. 2008 Feb 19;105(7):2693-8.
doi: 10.1073/pnas.0708452105. Epub 2008 Feb 7.

A Drosophila model for LRRK2-linked parkinsonism

Zhaohui Liu  1 , Xiaoyue Wang, Yi Yu, Xueping Li, Tao Wang, Haibing Jiang, Qiuting Ren, Yuchen Jiao, Akira Sawa, Timothy Moran, Christopher A Ross, Craig Montell, Wanli W Smith
Affiliations

A Drosophila model for LRRK2-linked parkinsonism

Zhaohui Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in the leucine-rich repeat kinase (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD) with pleiomorphic pathology. Previously, we and others found that expression of mutant LRRK2 causes neuronal degeneration in cell culture. Here we used the GAL4/UAS system to generate transgenic Drosophila expressing either wild-type human LRRK2 or LRRK2-G2019S, the most common mutation associated with PD. Expression of either wild-type human LRRK2 or LRRK2-G2019S in the photoreceptor cells caused retinal degeneration. Expression of LRRK2 or LRRK2-G2019S in neurons produced adult-onset selective loss of dopaminergic neurons, locomotor dysfunction, and early mortality. Expression of mutant G2019S-LRRK2 caused a more severe parkinsonism-like phenotype than expression of equivalent levels of wild-type LRRK2. Treatment with l-DOPA improved mutant LRRK2-induced locomotor impairment but did not prevent the loss of tyrosine hydroxylase-positive neurons. To our knowledge, this is the first in vivo"gain-of-function" model which recapitulates several key features of LRRK2-linked human parkinsonism. These flies may provide a useful model for studying LRRK2-linked pathogenesis and for future therapeutic screens for PD intervention.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
LRRK2 induced retinal degeneration. (A) Expression of Flag-LRRK2 and Flag-LRRK2-G2019S in photoreceptor cells. Fly head extracts prepared from the indicated transgenic flies were fractionated and subjected to Western blot analysis using anti-Flag antibodies. (B) Time course of photoreceptor degeneration determined by the optical neutralization technique. Each data point was based on examination of ≥90 ommatidia from at least six flies. Statistically significant differences between control and LRRK2 or LRRK2-G2019S transgenic flies are indicated: *, P < 0.05 by ANOVA. (C) Ommatidia of 5-week-old flies examined by transmission electron microscopy.
Fig. 2.
Fig. 2.
Expression of LRRK2 protein by ddc-GAL4 driver caused locomotor dysfunction. (A) Expression of LRRK2 proteins in flies containing the ddc-GAL4 in combination with the UAS-LRRK2 transgenes. Head extracts from the indicated fly stocks were subjected to Western blot analysis by using anti-Flag antibodies [20 μg of protein per lane, except for the ddc-GAL4;LRRK2–4 (10 μg)]. (B) Survival curves of flies expressing either LRRK2 or LRRK2-G2019S (n = 50). (C) Cohorts of 60 flies from each genotype were subjected to climbing assays weekly. Statistically significant differences between the control and LRRK2 transgenic lines (except the 4 week data) are indicated: *, P < 0.05 by ANOVA. Statistically significant differences between LRRK2-1 and G2019S-2 flies are indicated: #, P < 0.05 by ANOVA. (D) Cohorts of 20 flies from each genotype at 5 weeks of age were subjected to the actometer to measure locomotor activity. Shown are representative data from three separate experiments. (E) Flies at 5 weeks of age were untreated or were treated with 1 mM l-DOPA for 10 days, then subjected to climbing assays. Statistically significant differences between untreated ddc-GAL4;G2019S-2 flies are indicated: *, P < 0.05 by ANOVA.
Fig. 3.
Fig. 3.
Expression of LRRK2 protein by ddc-GAL4 driver induced loss of TH-positive DA neurons. (A) Diagram of DA and 5-HT neuron clusters in the medial and lateral areas of the adult fly brain as in previous publications (35, 36). (Left) Five clusters: PPM1 (unpaired), PPM2 (paired), PPM3 (paired; protocerebral posterior medial), and PPL1 and PPL2 (paired; protocerebral posterolateral) on the posterior side. (Center) Two DA clusters: PAL (protocerebral anterolateral) and PAM (paired anterolateral medial) on the anterior side. (Right) Five distinct 5-HT neuronal clusters (SP1, SP2, LP1, LP2, and IP) in the two brain hemispheres. (B–D) Dissected whole brains were subjected to anti-TH immunofluorescent staining. (B) Quantitation of TH-positive neurons in PPM1/2 clusters in transgenic flies of the indicated ages. (C) Average numbers of TH-positive neurons per DA cluster in 5-week-old flies of the indicated genotypes. (D) Representative images of anti-TH staining in PPM1 and PPM2 clusters from 5-week-old flies of the indicated genotypes. Statistically significant differences between the control and all LRRK2 transgenic lines are indicated: *, P < 0.05 by ANOVA. Statistically significant differences between LRRK2-1 and G2019S-2 flies are indicated: #, P < 0.05 by ANOVA.
Fig. 4.
Fig. 4.
Expression of LRRK2 by elav-GAL4 driver caused late-onset locomotor impairment. (A and B) Expression of CG5483 and human LRRK2 in various types of fly brain tissues. Total RNA was prepared from fly brain tissues, and cDNA was generated. Semiquantitative RT-PCR was performed by using primers for CG5483 and human LRRK2 to assess mRNA levels. (A) Representative image of RT-PCR products. (B) Quantitative analysis of relative mRNA levels of CG5483 and human LRRK2. *, P < 0.05 versus UAS-LRRK2 by ANOVA. (C) LRRK2 autophosphorylation (kinase) analysis of various fly head homogenates. Anti-Flag-LRRK2 immunoprecipitated samples from fly head homogenates were incubated with [γ-32P]ATP, subjected to SDS/PAGE, and blotted onto PVDF membranes. The samples were then imaged by using a phosphoimaging system. The incorporation of [γ-32P]ATP into LRRK2 protein increased by ≈2.8-fold in elav-GAL4;G2019S-2 flies compared with elav-GAL4;LRRK2-1 flies. Shown are representative images from three independent experiments. (D) Cohorts of 60 flies from each genotype were subjected to climbing assays weekly. Statistically significant differences between the control and all LRRK2 transgenic lines are indicated: *, P < 0.05 by ANOVA. Statistically significant differences between LRRK2-1 and G2019S-2 flies are indicated: #, P < 0.05 by ANOVA. (E) Survival curves of flies expressing either LRRK2 or LRRK2-G2019S (n = 50).
Fig. 5.
Fig. 5.
Expression of LRRK2 in all neurons caused selective loss of anti-TH-positive neurons. (A) Anti-elav whole-mount brain immunofluorescence of flies at 5 weeks after eclosion showed that expression of either LRRK2-1 or G2019S-2 did not significantly change the density of immunofluorescence. There was a slight but not significant decrease in anti-elav staining in LRRK2 transgenic flies. This could be due to loss of anti-TH-positive neurons. (B) Anti-5-HT whole-mount brain immunofluorescence of flies at 5 week after eclosion showed that expression of either LRRK2-1 or G2019S-2 did not cause loss of 5-HT-positive neurons. (C) Anti-TH whole-mount brain immunofluorescence of flies at 5 weeks after eclosion showed that expression of G2019S induced loss of TH-positive neurons. (D) Representative images of whole-mount brain sections of flies 5 weeks after eclosion. (Left) Anti-elav staining brain section. (Center) SP1, SP2, and IP 5-HT neuronal clusters with anti-5-HT staining. (Right) PPM1 and PPM2 TH-stained DA neuronal clusters.
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