title: Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease.
abstract: Pantothenate Kinase Associated Neurodegeneration (PKAN) is an autosomal recessive disorder with mutations in the pantothenate kinase 2 gene (PANK2), encoding an essential enzyme for Coenzyme A (CoA) biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. We exploited the zebrafish model to study the role played by the pank2 gene during embryonic development and get new insight into PKAN pathogenesis. The zebrafish orthologue of hPANK2 lies on chromosome 13, is a maternal gene expressed in all development stages and, in adult animals, is highly abundant in CNS, dorsal aorta and caudal vein. The injection of a splice-inhibiting morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the heart region and caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized the CNS phenotype by studying the expression pattern of wnt1 and neurog1 neural markers and by use of the Tg(neurod:EGFP/sox10:dsRed) transgenic line. The results evidenced that downregulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS (telencephalon). Whole-mount in situ hybridization analysis of the endothelial markers cadherin-5 and fli1a, and use of Tg(fli1a:EGFP/gata1a:dsRed) transgenic line, confirmed the essential role of pank2 in the formation of the vascular system. The specificity of the morpholino-induced phenotype was proved by the restoration of a normal development in a high percentage of embryos co-injected with pank2 mRNA. Also, addition of pantethine or CoA, but not of vitamin B5, to pank2 morpholino-injected embryos rescued the phenotype with high efficiency. The zebrafish model indicates the relevance of pank2 activity and CoA homeostasis for normal neuronal development and functioning and provides evidence of an unsuspected role for this enzyme and its product in vascular development.
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy. Electronic address: daniela.zizioli@unibs.it.
authors.forename: Daniela
authors.initials: D
authors.lastname: Zizioli
authors.affiliation: Department of Biology, University of Padova, via U. Bassi 58/B, 35121 Padova, Italy.
authors.forename: Natascia
authors.initials: N
authors.lastname: Tiso
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.
authors.forename: Adele
authors.initials: A
authors.lastname: Guglielmi
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Saraceno
authors.affiliation: Department of Biology, University of Padova, via U. Bassi 58/B, 35121 Padova, Italy.
authors.forename: Giorgia
authors.initials: G
authors.lastname: Busolin
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Giuliani
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.
authors.forename: Deepak
authors.initials: D
authors.lastname: Khatri
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Monti
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Borsani
authors.affiliation: Department of Biology, University of Padova, via U. Bassi 58/B, 35121 Padova, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Argenton
authors.affiliation: Department of Molecular and Translational Medicine, University of Brescia, viale Europa 11, 25123 Brescia, Italy; Clinical Chemistry Laboratory, Spedali Civili Hospital, 25123 Brescia, Italy. Electronic address: dario.finazzi@unibs.it.
authors.forename: Dario
authors.initials: D
authors.lastname: Finazzi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D019556:COS Cells; D002319:Cardiovascular System / Q000201:enzymology* / Q000254:growth & development* / Q000473:pathology; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D055785:Gene Knockdown Techniques; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009420:Nervous System / Q000201:enzymology* / Q000254:growth & development* / Q000473:pathology; D006211:Pantothenate Kinase-Associated Neurodegeneration / Q000473:pathology / Q000503:physiopathology; D010641:Phenotype; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015027:Zebrafish
chemical_list: D017853:Phosphotransferases (Alcohol Group Acceptor); C023088:pantothenate kinase
keywords: Coenzyme A; Morpholino; NBIA; Neurodegeneration; Pank2; Pantethine; Zebrafish