Knock-down of pantothenate kinase 2 severely affects the development of the nervous and vascular system in zebrafish, providing new insights into PKAN disease

Daniela Zizioli,Natascia Tiso,Adele Guglielmi,Claudia Saraceno,Giorgia Busolin,Roberta Giuliani,Deepak Khatri,Eugenio Monti,Giuseppe Borsani,Francesco Argenton,Dario Finazzi

doi:10.1016/j.nbd.2015.10.010

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.

Highlights

Pantothenate Kinase-Associated Neurodegeneration (PKAN, OMIM ID: 234200) is the most common form of a group of inherited neurological disorders named Neurodegeneration with Brain Iron Accumulation (NBIA) (Hayflick et al, 2003; Levi and Finazzi, 2014)
A phylogenetic analysis, carried out including all the pantothenic acid kinases identified in the four vertebrates considered for the multiple sequence alignment, shows that the D. rerio protein under investigation correctly segregates in the pantothenate kinase 2 gene (PANK2) clade (Fig. S1C)
Phenotype with hydrocephalus and edema of heart and tail, the vast majority (82%) of the embryos (n = 87/105 out of 3 microinjection) treated with Coenzyme A (CoA) had a normal phenotype, similar to that of STMO-injected embryos (Fig. 5). These results suggest that downregulation of pank2 can lead to a shortage of CoA in specific cells and tissues in zebrafish embryos, that can be compensated by metabolites entering the CoA biosynthetic pathway downstream of panthotenate phosphorylation

Summary

Introduction

Pantothenate Kinase-Associated Neurodegeneration (PKAN, OMIM ID: 234200) is the most common form of a group of inherited neurological disorders named Neurodegeneration with Brain Iron Accumulation (NBIA) (Hayflick et al, 2003; Levi and Finazzi, 2014). A growing body of evidence indicates that perturbation of CoA homeostasis is the key factor in disease development, and the identification of mutations in another enzyme of this biochemical pathway, COASY, in two patients with NBIA further strengthen this notion (Dusi et al, 2014) This is evident in the Drosophila model of the disease, where the reduced levels of expression of the only dPANK/Fbl enzyme results in reduced CoA levels, locomotor dysfunction and neurodegeneration with severe morphological alterations of mitochondria (Bosveld et al, 2008; Rana et al, 2010). We describe an in-depth morphofunctional characterization of the gene in zebrafish and provide functional insights of potential relevance for PKAN pathology in humans

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