The increased iron deposition is a hallmark of many neurodegenerative diseases, but its pathogenic role is still unclear. A strong link between iron and neurodegeneration is evident in a set of heterogeneous neurological disorders, known as Neurodegeneration with Brain Iron Accumulation (NBIA). The most common form of inherited NBIA is associated with mutations in the human PANK2 gene (PKAN disease). Pank2 is the rate-limiting enzyme in CoA biosynthesis and its down-regulation in mammalian cells leads to perturbation of cellular iron homeostasis. In our work we have explored the Pank2 biological function in zebrafish (Danio rerio), proposing this system as an important new tool for the study of PKAN disease. The zebrafish Pank2 protein shows 65% identity with the human ortholog. By qRT-PCR analysis on total RNA from embryos and adult tissues we have found that the expression of pank2 transcripts is detectable in embryos from early stages to 72 hours post-fertilization, and that the brain is the tissue with the highest expression level of pank2. The whole-mount in situ hybridization (WISH) technique confirms the qRT-PCR results, showing high pank2 expression in different brain structures, in the main vessels and in the venous plexus. The microinjection of pank2-specific morpholino oligos results in a clear-cut phenotype, with perturbation of CNS structures and vascular system development, suggesting the relevance of pank2 expression for the normal nervous and vascular developmental process in zebrafish. Both the co-injection of pank2 mRNA and the addition of pantethine 30 mM at the gastrula developmental stage can restore the wild type phenotype with high efficiency. The effects induced in the CNS and vascular structures have been characterized by WISH with different neuronal and vascular markers and by injecting pank2 morpholino oligos in different transgenic lines. The results indicate a clear effect on the development of the forebrain, where also the nuclei corresponding to the human globus pallidus are located. The vascular arborization is also drastically perturbed, with severe fenestration of the main vessels and reduced connections of the inter-somitic vessels. Altogether these data indicate that the transient down-regulation of pank2 gene expression in zebrafish represents an interesting model of PKAN disease, potentially amenable for high-throughput screening of molecules with therapeutic potential.

The down-regulation of pank2 gene in zebrafish as a model of Pantothenate Kinase Associated Neurodegeneration

TISO, NATASCIA;BUSOLIN, GIORGIA;ARGENTON, FRANCESCO;
2016

Abstract

The increased iron deposition is a hallmark of many neurodegenerative diseases, but its pathogenic role is still unclear. A strong link between iron and neurodegeneration is evident in a set of heterogeneous neurological disorders, known as Neurodegeneration with Brain Iron Accumulation (NBIA). The most common form of inherited NBIA is associated with mutations in the human PANK2 gene (PKAN disease). Pank2 is the rate-limiting enzyme in CoA biosynthesis and its down-regulation in mammalian cells leads to perturbation of cellular iron homeostasis. In our work we have explored the Pank2 biological function in zebrafish (Danio rerio), proposing this system as an important new tool for the study of PKAN disease. The zebrafish Pank2 protein shows 65% identity with the human ortholog. By qRT-PCR analysis on total RNA from embryos and adult tissues we have found that the expression of pank2 transcripts is detectable in embryos from early stages to 72 hours post-fertilization, and that the brain is the tissue with the highest expression level of pank2. The whole-mount in situ hybridization (WISH) technique confirms the qRT-PCR results, showing high pank2 expression in different brain structures, in the main vessels and in the venous plexus. The microinjection of pank2-specific morpholino oligos results in a clear-cut phenotype, with perturbation of CNS structures and vascular system development, suggesting the relevance of pank2 expression for the normal nervous and vascular developmental process in zebrafish. Both the co-injection of pank2 mRNA and the addition of pantethine 30 mM at the gastrula developmental stage can restore the wild type phenotype with high efficiency. The effects induced in the CNS and vascular structures have been characterized by WISH with different neuronal and vascular markers and by injecting pank2 morpholino oligos in different transgenic lines. The results indicate a clear effect on the development of the forebrain, where also the nuclei corresponding to the human globus pallidus are located. The vascular arborization is also drastically perturbed, with severe fenestration of the main vessels and reduced connections of the inter-somitic vessels. Altogether these data indicate that the transient down-regulation of pank2 gene expression in zebrafish represents an interesting model of PKAN disease, potentially amenable for high-throughput screening of molecules with therapeutic potential.
The 12th International Conference on Zebrafish Development and Genetics
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3208717
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