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. In patients, these mutations lead to neurodegeneration with brain iron accumulation, however, the underlying pathogenic mechanism is poorly understood yet. 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. Interestingly few mutations led to complete abrogation of enzymatic activity, while others had only a minor effect. We recently analyzed the role of pank2 in zebrafish during embryonal development. Specific downregulation of pank2 using morpholino resulted in significant alteration of neuronal and vascular development. 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 fishTg(fli1a:EGFP-gata1a:DsRed)overexpressing the same mutant proteins.No significant CNS alteration was observed in embryos microinjected with different mRNAs, but better details will come by using the transgenic line Tg(neurod:EGFP). 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. Measurement of CoA in each type of embryos is undergoing using a LC-MS method. This work could provide relevant information about PKAN pathogenesis and the exclusive association of PANK2 defects with lack of adequate level of intracellular CoA.

Overexpression of human mutant PANK2 mRNAs in zebrafish embryos to gain insight about PKAN pathogenesis

ZIZIOLI, DANIELA;TISO, NATASCIA;FACCHINELLO, NICOLA;
2017

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. In patients, these mutations lead to neurodegeneration with brain iron accumulation, however, the underlying pathogenic mechanism is poorly understood yet. 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. Interestingly few mutations led to complete abrogation of enzymatic activity, while others had only a minor effect. We recently analyzed the role of pank2 in zebrafish during embryonal development. Specific downregulation of pank2 using morpholino resulted in significant alteration of neuronal and vascular development. 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 fishTg(fli1a:EGFP-gata1a:DsRed)overexpressing the same mutant proteins.No significant CNS alteration was observed in embryos microinjected with different mRNAs, but better details will come by using the transgenic line Tg(neurod:EGFP). 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. Measurement of CoA in each type of embryos is undergoing using a LC-MS method. This work could provide relevant information about PKAN pathogenesis and the exclusive association of PANK2 defects with lack of adequate level of intracellular CoA.
NBIA 2017 – 6th International Symposium on NBIA and Related Disorders
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3228319
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