LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation

Abstract

Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of β-synuclein, PARK13, and SOD1 and causes β-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.

Loss oflrrk2impairs dopamine catabolism, cell proliferation, and neuronal regeneration in the zebrafish brain

“…Second, we analyzed the maternal-zygotic phenotype to circumvent potential maternal confounding effects. In contrast with published MO-induced phenotypes (22,24), we demonstrate that zebrafish lrrk2 is a resilient locus (Figure 2-4). In particular, mzLrrk2 larvae initially display reduced CA neurons and a combination of distinct motor signatures interpretable as hypokinesia that resolve spontaneously during further development.…”

“…To exclude any effect from the existing maternal wild-type lrrk2 transcripts (Supplemental Figure S2a), F2 fish were further incrossed to obtain homozygous mutants that developed from homozygous mothers (maternal-zygotic homozygous tud113 mutants, henceforth referred to as "mzLrrk2") and wild-type (wt) control lines. In striking contrast with published MO-induced phenotypes (22,24), mzLrrk2 individuals develop normally, are viable, and reach sexual maturity at the same age as controls, with both females and males being fertile. During zebrafish development, lrrk2 expression is ubiquitous until 24 hours post-fertilization (hpf), then gradually restricts to the head ( Supplementary Figure 1).…”

“…Using TUNEL assay, we measured a threefold increase of the cell death rate in mzLrrk2 brains at 5 dpf (anterior: P=0.0061; middle: P<0.0001; Figure 2a, a'), but not at 10 dpf (Figure 2b, b'). Unlike previous studies (22,24), no clear sign of neuronal loss was evident after HuC/D staining at either 5 or 10 dpf (Figure 2c Neuroinflammation is a concurring factor in many neurodegenerative conditions, including PD (30). Increasing evidence implicates LRRK2 in microglia function (31).…”

“…To study the gene function of zebrafish lrrk2 in vivo and potentially solve the controversy arising from previous MO studies (22)(23)(24), we removed the lrrk2 locus containing the entire ORF. This approach has two main advantages.…”

Lrrk2 modulation of Wnt signaling during zebrafish development

“…Loss of dopaminergic neurons is a defining feature of PD (Poewe et al., 2017). Previous Lrrk2 knockdown studies in zebrafish have come to opposing conclusions on the requirement for Lrrk2 in dopaminergic neuron specification (Prabhudesai et al., 2016; Ren et al., 2011; Sheng et al., 2010). To assess the requirement for Lrrk2 in early dopaminergic neuron development in our lrrk2 mutants, we performed whole mount RNA in situ hybridization using two markers of dopaminergic neurons, tyrosine hydroxylase ( th1) and d‐alanine aminotransferase ( dat) at 72 hpf on the lrrk2 sbu304/sbu304 mutants (Figure 3 and Figure S2), which are predicted to disrupt all of the key domains.…”