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.

Down-regulation of coasy, the gene associated with NBIA-VI, reduces Bmp signaling, perturbs dorso-ventral patterning and alters neuronal development in zebrafish

“…Down-regulation of pank2 in zebrafish embryos resulted in significant perturbation of CNS and vasculature structures13. We were interested in verifying the possible overlap between these two types of morphants that affect the same biochemical pathway.…”

“…The simplest interpretation of existing evidences links defects in PANK2 and COASY with shortage of cellular CoA that, in turn, acts as initial trigger of a cascade of events culminating with neural death in specific brain areas. This hypothesis is supported by the rescue capacity of pantethine and CoA documented both in cellular and animal models10111213. On the other hand, measurement of CoA concentrations in fibroblasts or blood samples from patients did not evidence reduced levels of the metabolite814.…”

“…These features are often associated with signs of lipid dyshomeostasis, implying possible perturbation in mitochondrial membrane remodeling, and disruption of iron balance1517. To gain new information about the functional connection between enzyme defects and pathology, we recently performed a thorough analysis of pank2 role during zebrafish embryonic development13. When we down-regulated larval pank2 expression by microinjection of a specific splice-inhibiting morpholino oligomer we observed a drastic reduction in neurogenin1 neurog1 and neurod1 positive neurons, particularly in telencephalon and midbrain, impaired development of main arteria, vein and intersegmental vessels and edema both in brain and at the tail plexus.…”

Inborn errors of coenzyme A metabolism and neurodegeneration

“…Morpholino‐mediated Pank2 down‐regulation causes abnormal development of central nervous system (CNS) and vascular structures. Significant oedema in the midbrain/hindbrain ventricle and a severe perturbation of brain morphology seem to be associated with a clear loss of neural cells primarily in telencephalon, the proposed fish’s brain area homologous to vertebrate pallidum …”

Genes relacionados con microftalmia y anoftalmia hereditarias

“…Subsecuentemente, los estudios funcionales, de expresión y en modelos animales han confirmado la participación de estos genes en las vías del desarrollo ocular normal 75,76 . La disponibilidad de nuevas tecnologías, como la secuenciación del genoma completo y la secuenciación de exoma completo, han aumentado de manera notable la capacidad de identificar nuevos genes relacionados con este tipo de anomalías hereditarias.…”