Loss-of function of pank2 gene in zebrafish as a model of pantothenate kinase associated neurodegeneration

We are developing zebrafish models of PKAN and COPAN, two types of Neurodegeneration with Brain Iron Accumulation disorders associated to mutations in genes involved in CoA biosynthesis. We describe, for the first time, the results obtained by loss-of-function of pank2 expression during the early stages of zebrafish development. The zebrafish pank2 gene is located on chromosome 13 and it is the ortholog of the human PANK2. When expressed in mammalian cells, the zebrafish protein localized in the cytosol and in the nucleus. WISH analysis showed the highest expression in the CNS, in the dorsal aorta, in the caudal vein and plexus at 24 and 48 hpf. The injected embryos with 1 pmol of splicing inhibiting morpholino showed a severe phenotype; more than 70% of injected embryos showed a poorly-defined brain morphology and hydrocephalus; edema was also present in the heart region and in the caudal plexus, where hemorrhages with reduction of blood circulation velocity were detected. We characterized more deeply the neural phenotype by studying the expression pattern of wnt1, neurod and ngn1 genes. We observed down-regulation of the three markers in morpholino-injected embryos, and these results were confirmed by using transgenic lines such as neurodGFP/sox10dsRED. Loss-of-function of pank2 severely impairs neuronal development, particularly in the forebrain (telencephalon). The WISH analysis using endothelial markers such as VE-cadherin and fli1, as well as the use of the fli1a:EGFP/gata1aDSRed transgenic line, confirmed the role of pank2 during vessels formation. The specificity of the observed phenotype was then verified by the co-injection of the morpholino and pank2 mRNA; the mRNA rescued toward a normal development in about 60% of injected embryos. Interestingly, the addiction of pantethine, a CoA precursor, in the fish-water at 5 hpf, rescued the phenotype in a large percentage of the injected embryos. In conclusion, the zebrafish model shows the relevance of pank2 activity and CoA homeostasis for normal neuronal development and function, and also indicates a new and unsuspected role for this protein and metabolite in vascular development.