PKAN is an autosomal recessive disorder with mutations in the PANK2 gene, encoding an essen- tial enzyme for CoA biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. More than 50 mutations have been identified in PKAN patients; the genotype/phenotype co-relation has been analyzed by studying the in vitro activity of recombinant mutant proteins. The zebrafish orthologue of human PANK2 lies on chromosome 13, is a maternal gene expressed in all devel- opment stages also it is highly abundant in CNS, dorsal aorta and caudal vein. The morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the 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 transgenic line. The results evidenced that down- regulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS. WISH analysis of the endothelial markers cadherin-5 and fli1a, and use of 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. We tried to extend this study by overexpressing in zebrafish embryos human PANK2 mRNAs carrying 4 different point mutations. Two point mutations (G219V and S471N) completely lack enzymatic activity whereas other two (T234A and T528M) do not show much changes. Microinjection of wild type PANK2 mRNA at 1 cell stage did not result in any morphological alteration, whereas the injection of mutant mRNAs led to the appearance of a graded phenotype, with frequent observation of curvy and thinner tail, hemorrhage in the tail and improper formation of the yolk. Tail hemorrhages were often observed in pank2 morphants. Surprisingly these alterations were milder in G219V and S471N mutants that lack enzymatic activity, while they were more significant in T234A and T528M. The alterations in vascular structure were also evident in transgenic fish overexpressing the same mutant proteins. It is known that mutations in PANK2 lead to deficient production of CoA, hence we tried to rescue the altered phenotype by providing CoA into the fish water. In preliminary experiments, we observed the significant recovery of tail and yolk structure, while hemorrhages were reduced but not completely absent, especially in T234A mutants. 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. References 1. Zizioli D et al., 2015 Oct 18, ýNeurobiol. Dis, doi: 10.1016/j.nbd.2015.10.010. 2. Khatri D et al., 2016 Nov 28, doi: 10.1038/srep37660

Knock-down of pank2 in zebrafish and overexpression of human PANK2 in zebrafish: insight for PKAN pathogenesis

ZIZIOLI, DANIELA;TISO, NATASCIA;GIANONCELLI, ALESSANDRA;
2017

Abstract

PKAN is an autosomal recessive disorder with mutations in the PANK2 gene, encoding an essen- tial enzyme for CoA biosynthesis. The molecular connection between defects in this enzyme and the neurodegenerative phenotype observed in PKAN patients is still poorly understood. More than 50 mutations have been identified in PKAN patients; the genotype/phenotype co-relation has been analyzed by studying the in vitro activity of recombinant mutant proteins. The zebrafish orthologue of human PANK2 lies on chromosome 13, is a maternal gene expressed in all devel- opment stages also it is highly abundant in CNS, dorsal aorta and caudal vein. The morpholino induced a clear phenotype with perturbed brain morphology and hydrocephalus; edema was present in the 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 transgenic line. The results evidenced that down- regulation of pank2 severely impairs neuronal development, particularly in the anterior part of CNS. WISH analysis of the endothelial markers cadherin-5 and fli1a, and use of 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. We tried to extend this study by overexpressing in zebrafish embryos human PANK2 mRNAs carrying 4 different point mutations. Two point mutations (G219V and S471N) completely lack enzymatic activity whereas other two (T234A and T528M) do not show much changes. Microinjection of wild type PANK2 mRNA at 1 cell stage did not result in any morphological alteration, whereas the injection of mutant mRNAs led to the appearance of a graded phenotype, with frequent observation of curvy and thinner tail, hemorrhage in the tail and improper formation of the yolk. Tail hemorrhages were often observed in pank2 morphants. Surprisingly these alterations were milder in G219V and S471N mutants that lack enzymatic activity, while they were more significant in T234A and T528M. The alterations in vascular structure were also evident in transgenic fish overexpressing the same mutant proteins. It is known that mutations in PANK2 lead to deficient production of CoA, hence we tried to rescue the altered phenotype by providing CoA into the fish water. In preliminary experiments, we observed the significant recovery of tail and yolk structure, while hemorrhages were reduced but not completely absent, especially in T234A mutants. 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. References 1. Zizioli D et al., 2015 Oct 18, ýNeurobiol. Dis, doi: 10.1016/j.nbd.2015.10.010. 2. Khatri D et al., 2016 Nov 28, doi: 10.1038/srep37660
10th European Zebrafish Meeting - Book of Abstracts
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