Deletion of the WD40 domain of LRRK2 in Zebrafish causes Parkinsonism-like loss of neurons and locomotive defect

Abstract

LRRK2 plays an important role in Parkinson's disease (PD), but its biological functions are largely unknown. Here, we cloned the homolog of human LRRK2, characterized its expression, and investigated its biological functions in zebrafish. The blockage of zebrafish LRRK2 (zLRRK2) protein by morpholinos caused embryonic lethality and severe developmental defects such as growth retardation and loss of neurons. In contrast, the deletion of the WD40 domain of zLRRK2 by morpholinos targeting splicing did not induce severe embryonic developmental defects; rather it caused Parkinsonism-like phenotypes, including loss of dopaminergic neurons in diencephalon and locomotion defects. These neurodegenerative and locomotion defects could be rescued by over-expressing zLRRK2 or hLRRK2 mRNA. The administration of L-dopa could also rescue the locomotion defects, but not the neurodegeneration. Taken together, our results demonstrate that zLRRK2 is an ortholog of hLRRK2 and that the deletion of WD40 domain of zLRRK2 provides a disease model for PD.

Figure 1. Expression profiling of lrrk2 in zebrafish.

(A) Quantitative RT–PCR analysis of zlrrk2 mRNA expression from 1 cell stage to 10 dpf. (B–D) Northern blot analysis (B), quantitative RT–PCR (C), and Western blot analyses (D) of zLRRK2 expression in gut, muscle, ovary, and brain of adult fish. (E) In situ hybridization of zlrrk2 mRNA at 24 hpf and 6 dpf stage. For 6 dpf stage, cryo-sectioning was performed in the position labeled as 1 and 2.

Figure 2. Conservation of amino acid sequences and functional domains of LRRK2 between zebrafish and human.

Figure 3. Phenotype of neuronal loss in LRRK2 ΔWD40 morphants.

(A) Western blot analysis at 2 dpf and 3 dpf confirming the knockdown effect of zLRRK2 wild-type protein expression as well as the decreased TH protein expression in WD40 deletion morphant. Adult brain protein was used as positive control, and the ATG morphant at 3 dpf was used as negative control. The quantified results are shown in Figure S2. (B) WISH analysis at 3 dpf showing that WD40 domain deletion caused decreased TH and DAT expressions in WD40 deletion morphant, but without significant brain developmental retardation. (C) Uninjected (left) and WD40 deleted zebrafish expressing Kaede under the deltaD promoter. While neuron numbers is not much changed at 18 somite stage, there are fewer neurons in the midbrain of these 6 dpf fish, as indicated by the arrowheads. ***P<0.001, n.s.: not significant (unpaired Student's t-test). (D) Cell apoptosis assay of uninjected (left) and WD40 delete zebrafish embryos.

Figure 4. Analysis by acetylated tubulin staining of embryos at 6 dpf.

Compared to the uninjected embryo, the WD40 deletion morphant has reduced and disorganized axon tracts in the midbrain of zebrafish. This phenotype could be rescued by over-expressing either zLRRK2 (WD40+zLRRK2) or hLRRK2 (WD40+hLRRK2) mRNA. **P<0.01, ***P<0.001 (unpaired Student's t-test).

Figure 5. WISH Analysis of the loss of TH+ cell in WD40 morphant and its rescue by over-expressing wild-type zLRRK2 or hLRRK2 as well as hLRRK2 mutant alleles.

(A) WISH analysis at 3 dpf shows the decreased TH expression in WD40 deletion morphant as well as the successful rescue by over-expressing either zLRRK2 or hLRRK2. The analysis also shows the decreased TH expression in the fish with over-expression of wild-type zLRRK2 and hLRRK2. This neurodegenerative phenotype could not be rescued by the treatment of L-dopa. (B) Quantification of rescue effect of zLRRK2 or hLRRK2 on TH+ cell loss in WD40 morphant. ***P<0.001 (unpaired Student's t-test). (C) Quantification of rescue effect of hG2019S or hG2385R on TH+ cell loss in WD40 morphant. **P<0.01, ***P<0.001 (unpaired Student's t-test).

Figure 6. Analysis of locomotive movement by measuring the swimming distance in 30 seconds.

The WD40 morphants swam much less distance than the wild-type fish. This locomotive defect could be rescued by over-expressing either zLRRK2 (WD40+zLRRK2) or hLRRK2 (WD40+hLRRK2) mRNA as well as by L-dopa treatment (WD40 + L-dopa). Each bar indicates the distance of an individual fish moved within 30 seconds. Each red dot (connected by red line) indicates the average distance of each of the five fish pools moved within 30 seconds, and the bar represents the SD of distance. *P<0.05, **P<0.01, n.s.: not significant (unpaired Student's t-test). It is noticeable that one of the ΔWD40 fish showed a very different phenotype from the rest. The distinct phenotype of this ‘outlier’ fish was likely due to either the failure or in-efficient knock-down effect by morpholino. The rescue effect by over-expressing hLRRK2 became statistically significant (P = 0.03) when the possible outlier of the ΔWD40 group (black arrow) is removed from the statistical analysis.