Overexpression of Human Mutant PANK2 Proteins Affects Development and Motor Behavior of Zebrafish Embryos

Abstract

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a genetic and early-onset neurodegenerative disorder characterized by iron accumulation in the basal ganglia. It is due to mutations in Pantothenate Kinase 2 (PANK2), an enzyme that catalyzes the phosphorylation of vitamin B5, first and essential step in coenzyme A (CoA) biosynthesis. Most likely, an unbalance of the neuronal levels of this important cofactor represents the initial trigger of the neurodegenerative process, yet a complete understanding of the connection between PANK2 malfunctioning and neuronal death is lacking. Most PKAN patients carry mutations in both alleles and a loss of function mechanism is proposed to explain the pathology. When PANK2 mutants were analyzed for stability, dimerization capacity, and enzymatic activity in vitro, many of them showed properties like the wild-type form. To further explore this aspect, we overexpressed the wild-type protein, two mutant forms with reduced kinase activity and two retaining the catalytic activity in zebrafish embryos and analyzed the morpho-functional consequences. While the wild-type protein had no effects, all mutant proteins generated phenotypes that partially resembled those observed in pank2 and coasy morphants and were rescued by CoA and vitamin B5 supplementation. The overexpression of PANK2 mutant forms appears to be associated with perturbation in CoA availability, irrespective of their catalytic activity.

Abnormal Vasculature Development in Zebrafish Embryos with Reduced Expression of Pantothenate Kinase 2 Gene

“…Knocking down pank2 expression by a specific morpholino led to perturbations of the neuronal development and vasculature formation. We found impaired angiogenesis in mutant embryos with PANK2 overexpression [7] and with downregulation of the coasy gene [6], and also in mammalian cells with PANK2 silencing [10], which confirmed the relevance of CoA homeostasis for these processes. Vasculature function was not assessed in other animal models of PKAN.…”

Zebrafish Larvae as a Behavioral Model in Neuropharmacology

“…During this period, zebrafish possess a plethora of behaviors which could be analyzed automatically or manually and quantitatively or qualitatively. The zebrafish behaviors have been exploited in diverse research areas such as ethology, toxicology, pharmacology, neuroscience, and genetics [39,40,41,42,43,44,45]. In neuropharmacology, the behavior repertoire of zebrafish larvae has been utilized in a multipurpose way—for identifying new drugs with central nervous system (CNS) effect, for repurposing of drugs with CNS effects, for identifying drugs for various psychiatric illnesses such as anxiety and mood disorders, for the study of drugs affecting sleep disorders, and for identifying the therapeutic and target specificity of newer compounds [19,46,47,48].…”

Coenzyme a Biochemistry: From Neurodevelopment to Neurodegeneration

“…The downregulation of pank2 expression in zebrafish altered the formation of the central nervous system [73]; embryos injected with a specific morpholino showed hydrocephalus and a significant reduction of neural markers in the brain, particularly in the basal telencephalon, which should contain regions homologous to the pallidum [74]. At the same time, severe perturbations of the main vessels and the vascular plexus in the tail were also frequent, suggesting the involvement of the enzyme in vasculature development; interestingly, the overexpression of mutant forms of the human PANK2 in zebrafish embryos induced similar vascular abnormalities [75] and silencing of the enzyme in human umbilical vein endothelial cells resulted in defective angiogenic properties [76]. This aspect has not been analysed in other experimental settings and awaits further investigation.…”