Dopaminergic Neuronal Loss and Dopamine-Dependent Locomotor Defects in Fbxo7-Deficient Zebrafish

<div><p>Recessive mutations in the <em>F-box only protein 7</em> gene (<em>FBXO7</em>) cause PARK15, a Mendelian form of early-onset, levodopa-responsive parkinsonism with severe loss of nigrostriatal dopaminergic neurons. However, the function of the protein encoded by <em>FBXO7</em>, and the pathogenesis of PARK15 remain unknown. No animal models of this disease exist. Here, we report the generation of a vertebrate model of PARK15 in zebrafish. We first show that the zebrafish Fbxo7 homolog protein (zFbxo7) is expressed abundantly in the normal zebrafish brain. Next, we used two zFbxo7-specific morpholinos (targeting protein translation and mRNA splicing, respectively), to knock down the zFbxo7 expression. The injection of either of these zFbxo7-specific morpholinos in the fish embryos induced a marked decrease in the zFbxo7 protein expression, and a range of developmental defects. Furthermore, whole-mount <em>in situ</em> mRNA hybridization showed abnormal patterning and significant decrease in the number of diencephalic <em>tyrosine hydroxylase</em>-expressing neurons, corresponding to the human nigrostriatal or ventral tegmental dopaminergic neurons. Of note, the number of the <em>dopamine transporter</em>-expressing neurons was much more severely depleted, suggesting dopaminergic dysfunctions earlier and larger than those due to neuronal loss. Last, the zFbxo7 morphants displayed severe locomotor disturbances (bradykinesia), which were dramatically improved by the dopaminergic agonist apomorphine. The severity of these morphological and behavioral abnormalities correlated with the severity of zFbxo7 protein deficiency. Moreover, the effects of the co-injection of zFbxo7- and p53-specific morpholinos were similar to those obtained with zFbxo7-specific morpholinos alone, supporting further the contention that the observed phenotypes were specifically due to the knock down of zFbxo7. In conclusion, this novel vertebrate model reproduces pathologic and behavioral hallmarks of human parkinsonism (dopaminergic neuronal loss and dopamine-dependent bradykinesia), representing therefore a valid tool for investigating the mechanisms of selective dopaminergic neuronal death, and screening for modifier genes and therapeutic compounds.</p> </div

<i>zFbxo7</i> knock down results in dopaminergic neuronal cell loss.

<p>The brain catecholaminergic neurons were visualized by whole-mount <i>in situ</i> hybridization using antisense RNA probes specific for <i>tyrosine hydroxylase</i> (<i>th</i>, panel A) or <i>dopamine transporter</i> (<i>dat</i>, panel C). Number of neurons were counted manually and normalized to the counts in wild type zebrafish (panels B and D). * <i>P</i><0.01</p

Off-target effects due to MO-induced p53 activation are not detected.

<p>(A) Representative images of zebrafish embryos treated with single MO injection (ATG-MO, SP-MO or P53-MO) or co-injection (ATG-MO/P53-MO or SP-MO/P53-MO). (B) Percentage of healthy phenotype, mild phenotype abnormalities, severe phenotype abnormalities and lethality among uninjected control (WT), single injected morphants and co-injected morphants.</p

<i>zFbxo7</i> knock down results in locomotor defects, which are improved by apomorphine.

<p>The movements of WT zebrafish, ATG-MO-Mild and SP-MO-Mild morphants were recorded during three cycles of 10-minutes light/10-minutes darkness (periods of darkness are shown in grey). Compared with WT, morphants showed significantly decreased velocity in both light and dark phases (<i>P</i><0.01, panel A), which were significantly improved by treatment with apomorphine (<i>P</i><0.01 in the dark phase, panels B and C). Dom: domperidone. Apo: apomorphine.</p

<i>zFbxo7</i> knock down results in developmental defects.

<p>(A) Representative images of zebrafish wild type and morphants. Injection of ATG-MO or SP-MO induced a range of phenotypes, which were grouped in mild and severe, including curly tails (black arrowheads), heart edema and heart malformations (grey arrowheads). (B) Percentages of healthy phenotype, mild phenotype abnormalities, severe phenotype abnormalities and lethality among uninjected control (WT) and MOs-injected morphants. <i>zFbxo7</i> knock down results in decreased zFbxo7 protein expression</p

Characterization of the zFbxo7 protein in zebrafish.

<p>(A) Schematic representation of the zFbxo7 functional domains. The values underneath each domain indicate the amino acids identity between zFbxo7 and hFBXO7 (isoform1). Ubl: ubiquitin-like domain; FP: FBXO7/ PI31 domain; F-box: F-box motif; PRR: proline rich domain. (B) Western blot analysis of the zFbxo7 protein expression at different developmental stages. (C) Western blot analysis of zFbxo7 protein expression in different tissues of eight-month-old adult zebrafish. Actin was used as reference protein.(D) Immunostaining of the zFbxo7 protein in eight-month-old zebrafish brain areas. The zFbxo7 immunoreactivity is shown in brown, while the cell nuclei are counterstained in blue using hematoxylin. The following areas are shown: olfactory bulb (OB), diencephalon (Di), optic tectum (TeO), medulla oblongata (MO), habenula (Ha), and cerebellum (C). Scale bars: 100 µm.</p

<i>zFbxo7</i> knock down results in decreased zFbxo7 protein expression.

<p>(A) Western blot of the zFbxo7 protein at 72 hpf in uninjected control (WT) and MOs-injected morphants which showed mild or severe phenotype abnormalities. (B) Quantification of the zFbxo7 protein levels is shown in panel A (Odyssey software). Data were collected from three independent experiments, <i>P</i><0.01.</p

Phenotypic assays on wild-type and <i>fmr1</i> mutant embryos.

<p>A) Wild type and mutant embryos were analyzed using whole mount <i>in situ</i> hybridisation using probes against <i>dlx-2a</i>, <i>axial</i> and <i>islet-1</i>. B) The width of Meckel's cartilage was measured in wild type (n = 9) and MZ <i>fmr1</i> mutant (n = 11) embryos. The angle of this structure with regard to the anterior-posterior axis was also measured in wild-type (n = 6) and <i>fmr1</i> mutant (n = 9) embryos. Indicated errors represent SD. C) Neurite branching was measured on Rohon-Beard neurites using the monoclonal antibody zn-12. Plotted is the branching frequency per 1000 µm in both wild-type and MZ stop mutant embryos. In total n = 25 neurites (wild-type) and n = 28 neurites (MZfmr1) were traced in a total of 8 embryos of each genotype. Error bars represent SD.</p

Unsupervised hierarchical clustering of differentially abundant proteins (n = 263 proteins with >2 peptides) based on their MS-peak intensity values.

<p>Each exosome sample was analyzed in triplicate. Results were mean centered and log-transformed. Relative protein abundance is colored-coded with red corresponding to a relatively high abundance, green r corresponding to a relatively low abundance, and grey indicating missing abundance values.</p

XPO1, FASN and PDCD6IP abundance by immunohistochemistry on normal adjacent prostate (NAP), low-grade prostate cancer (Gleason score 3+3 = 6) and high grade prostate cancer (Gleason score 4+5 = 9).

<p>Representative pictures of the staining from 2 independent samples per group.</p