Disruption of LRRK2 Does Not Cause Specific Loss of Dopaminergic Neurons in Zebrafish

Mutations in LRRK2 are genetically linked to Parkinson's disease (PD) but its normal biological function is largely unknown. Sheng et al. recently reported that deletion of the WD40 domain of LRRK2 in zebrafish specifically causes PD-like loss of neurons and behavior defect. However, our similar early study and recent confirming experiments using the same reagents reported by Sheng et al. failed to reproduce the phenotype of the loss of dopaminergic neurons, although the mRNA of LRRK2 was molecularly disrupted. Our study suggests that function of LRRK2 and its usefulness to generate zebrafish PD model needs further evaluation.


Introduction
Parkinson's disease (PD) is a common neurodegenerative disorder affecting approximately 1% of the population over the age of 50 [1]. The primary symptoms of PD are movement dysfunctions, including tremor, rigidity, bradykinesia, and postural instability [2]. The pathologic hallmarks of PD are loss of dopaminergic (DA) neurons in the substantia nigra (SN) and the presence of Lewy bodies in the brain. Although most PD patients are idiopathic, 5-10% of PD patients are diagnosed to be linked to certain gene mutations, such as a-synuclein, UCHL1, LRRK2 (Leucine-rich repeat kinase 2), PINK1, Parkin, DJ-1, and ATP13A2 [3]. Among these genes, LRRK2 represents the most prevalent genetic cause of autosomal-dominant PD [4,5,6,7].
Human LRRK2 encodes a huge protein of 2527 amino acid and contains several functional domains including ARM (Armadillo), ANK (Ankyrin repeat), LRR (Leucine rich repeat), Roc (Ras of complex proteins, GTPase), COR (C-terminal of Roc), MAPKKK (Mitogen activated kinase kinase kinase), and WD40 from the N-terminus to the C-terminus. Over 40 point mutations have been identified in LRRK2, covering all of the functional domains, but proven pathogenic mutations appear concentrated in the GTPase and kinase domains [3,8]. The most common pathogenic mutation is G2019S in the kinase domain, which is identified in ,1% sporadic PD patients and ,4% familial PD patients [9]. Overexpression of pathogenic variants of LRRK2 is toxic in cultured neuronal cells [10,11]. In vitro kinase activity assay using moesin as substrate showed mutation G2019S increased the kinase activity of LRRK2, implying that the hyper kinase activity of LRRK2 is the cause of PD [12]. In transgenic mice, overexpression of mutant LRRK2 R1441G resulted in age-dependent and levodopa-responsive slowness of movement while overexpression of wild type LRRK2 did not cause typical symptom of PD [13]. In another study, overexpression of LRRK2 G2019S caused decreases in striatal dopamine content, release, and uptake [14]. However, the most characteristic feature of PD, loss of DA neurons, was not observed in these transgenic mice. Also, transgenic overexpession does not address the loss of function issue of LRRK2, which is critical for providing information of its normal biological function.
Zebrafish is a well established animal model for studying human diseases and has been used to investigate PD [15]. In addition, zebrafish embryos are susceptible to the treatment by the classic dopaminergic neurotoxin MPTP (1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine), which causes loss of DA neurons in zebrafish embryonic diencephalon, mimicking the key feature of PD [16,17]. Recently, Sheng et al. reported a functional study of LRRK2 in zebrafish, showing that deletion of the WD40 domain of zebrafish LRRK2 produced typical PD phenotype including specific loss of DA neurons and locomotive defect without overall developmental defect [18]. We have also been investigating LRRK2 function in zebrafish prior to Sheng et al. 's publication but were not able to observe the reported phenotype. In more recent studies, we further confirmed that the DA neurons appear normal in embryos with depletion of LRRK2 mRNA by injection of the exact same morpholino oligos (MOs) reported by Sheng et al. Our studies suggest that function of LRRK2 and its usefulness to generate zebrafish PD model needs further evaluation.

Design and validation of MOs targeting LRRK2
Bioinformatics analysis of genome sequence revealed that zebrafish contains a single copy of highly conserved LRRK2 homolog (Ensembl ENSDARG00000006169) that has all of the functional domains [18]. We observed similar expression pattern of LRRK2 as reported by Sheng et al. To study the function of LRRK2 in zebrafish, three MOs (MO45, MO50, and MO51IE') were designed to disrupt the splicing of kinase and WD40 domains of LRRK2 (Fig. 1). To determine whether these three MOs did not produce the reported phenotype due to the difference of targeting sites, the two exact RNA splice-blocking MOs (IE and EI) used by Sheng et al. were tested and re-named as MO51IE and MO51EI, respectively in this paper. Our MO51IE' is one nucleotide upstream to MO51IE and to make comparison concise, the data of MO51IE' is not shown.
The efficiency of these MOs was evaluated by RT-PCR with primers flanking the MO target sites at 3 dpf (days post fertilization) embryos ( Fig. 1). At a dose of 8 ng per embryo, all MOs were able to block the normal splicing of zebrafish LRRK2, leading to exon deletion ( Fig. 2A, bands a to c). Co-injection of MO51IE+MO51EI resulted in dramatic decrease of wild type LRRK2 transcript ( Fig. 2A, band

Knockdown of LRRK2 does not cause loss of DA neurons in zebrafish embryo
The MOs were injected into zebrafish embryos at a series of doses (Table 1). Most morphants developed 4-hour slower than un-injected control embryos without notable morphological defect at the dose of 8 ng per embryo or lower. We used 8 ng per embryo for experiments of this report since this concentration produced efficient block of LRRK2 mRNA splicing, as shown in Figure 2. At 3 dpf, in situ hybridization with dat (dopamine transporter) probe was performed to detected DA neurons [19]. For each of these MOs or MO combination, we did not observe loss of DA neurons in morphants (Fig. 3). Although MO50 and MO51IE +MO51EI caused some irregular patterns of DA neurons in diencephalons, specific loss of DA neurons was still not observed ( Fig. 3J, K, L and M).
In our studies of PD with zebrafish, we prefer using dat as probe to detect DA neurons since it is the most specific marker for DA neurons. However, Sheng et al. mainly used th (tyrosine hydroxylase) as probe to detect DA neurons in their report. To be consistent with their studies, we performed the same in situ hybridization using th probe and, again, no loss of DA neurons was observed in morphants (Fig. 4). There should be no difference between dat and th in detecting DA neurons because both of them are well established marker genes for PD studies [19,20].
We also examined the locomotor phenotype of morphants and used their un-injected siblings as control. Locomotor experiments included tactile response test at 3 dpf and swimming ability assay at 6 dpf. Consistent with our in situ hybridization result with either dat or th probe, there were not obvious differences between control and morphant (Fig. S2).
Loss of function study of LRRK2 homologs in C. elegans, Drosophila, and mouse has been carried out. In C. elegans, lrk-1 (LRRK2 homolog) regulates axonal-dendritic polarity of SV proteins, stress response and neurite outgrowth, but does not affect DA neurons [21,22]. In Drosophila, two groups reported contrasting findings: Lee et al. reported that loss of LRRK (LRRK2 homolog) induced severe reduction of DA neurons [23], while Wang et al. reported that the LRRK mutant fruit fly developed normally without changing the number and pattern of DA neurons [24]. In LRRK2 knockout mouse, the dopaminergic system appeared intact in both young and aged mice. Furthermore, there was no significant difference in the susceptibility of LRRK2 knockout and wild type mice to MPTP [25]. Collectively, the findings in model animals suggest that LRRK2 plays a very limited role in the development or maintenance of DA neurons [7]. In conclusion, our study indicates that disruption of LRRK2 in zebrafish did not lead to loss of DA neurons. Considering the limitations of knockdown with antisense morpholino oligo and the importance of LRRK2 gene, to obtain a more solid conclusion of the function of LRRK2 in embryonic development and generation of DA neurons, we would like to employ engineered zinc-finger nucleases to inactivate LRRK2 in zebrafish in future study.

Zebrafish maintenance
Wild type AB was maintained in a circulating aquaculture system according to standard described in The Zebrafish Book [26]. Embryos were incubated at 28.5uC and staged according to the description by Kimmel et al [27]. At 24 hours post fertilization, 1-phenyl-2-thiourea (PTU, Sigma-Aldrich, St. Louis, MO,) was added to a final concentration of 0.003% to prevent the production of pigment. This zebrafish study was approved by Peking University Shenzhen Graduate School (09316).     length of liquid injected (20 times) into the glass capillary, the volume per injection was thereby calculated, then the corresponding amount of MOs was also calculated. Injected embryos (morphant) were cultured in fish water at 28.5uC and allowed to grow up to 6 days.

Quantitative PCR
30 embryos of each group at 3 dpf were harvested. Total RNA was isolated with RNAqueous H -4PCR Kit (Ambion, Austin, TX) and cDNAs were generated with PrimeScript TM RT reagent Kit (Takara, Dalian, China). For LRRK2, the primers were as follows: 59GACTCCGAGGCGATACAG39 (forward, 778-795) and 59CAAGGGCACTCAGACAGG39 (reverse, 935-952); for beta-actin, the primers were as follows: 59 GCCGTGACCT-GACTGACTACCT39 (forward) and 59 CGCAAGATTCCA-TACCCAAGA39 (reverse). Quantitative PCR was carried out on a 7300 real time PCR system (Applied Biosystems, Carlsbad, CA) with SYBR H PrimeScript H RT-PCR Kit (Takara, Dalian, China). Presented data were shown as mean6S.E.M of biological triplicates from at least two independent experiments and were presented as fold changes in relative expression as compared with control after being normalized to beta-actin. The relative fold changes were calculated by a comparative C t method [28].

Whole mount in situ hybridization and counting DA neurons
Digoxigenin-labeled anti-sense RNA probes were generated in vitro by using the zebrafish dat and th cDNA as templates with RNA polymerase (Promega, Madison, WI). Whole-mount RNA in situ hybridizations were performed essentially as described by Westerfield [26]. After in situ hybridization, transfer embryos into glycerol and equilibrate for 10 min. Then put it on a slide and flatten it softly with a cover slip to disperse the neurons (Fig. S1). The number of dat or th positive neurons in the diencephalon region was counted manually by two persons blinded to the knockdown of LRRK2.

Imaging
Pictures of zebrafish embryos were taken with AxioImager A1 microscope and AxioCam digital camera (Zeiss, Oberkochen, Germany), and edited with Photoshop 7.0 (Adobe systems, San Jose, CA).

Behavior assay
Tactile response assay of 3 dpf embryo was modified from Xi et al., 2010 [29]. Embryos in a 10 cm-diameter petri dish were put in the centre of a 3.5 cm field of a dissecting microscope (Zeiss, Oberkochen, Germany). Gently touched the tail of embryo with a forcep and recorded manually the times of touch to drive the embryo out of the field. Swimming ability assay of 6 dpf embryo was modified from Levin and Cerutti, 2009[30]. An embryo was put in a well (diameter = 3.5 cm) containing 3 ml fish water and being divided equally to 8 parts, accommodated for 10 min, then manually recorded the times of segment crossing to different parts by an embryo within 5min.

Statistical analysis
All statistical analysis was performed with SPSS. The comparisons of differences between control and morphant groups were performed using one-way ANOVA. When the P value was less than 0.05, a Tukey HSD pairwise comparison post hoc test was conducted to determine the significance of difference between control and morphant groups. Error bars represented 6 S.E.M. The number of used embryo was shown in the corresponding figure legend. All experiments were independently repeated at least three times. P values less than 0.05 were considered to be significant.