Abstract
Neonatal epileptic encephalopathy (NEE), as a result of pyridoxine 5′-phosphate oxidase (PNPO) deficiency, is a rare neural disorder characterized by intractable seizures and usually leads to early infant death. The clinical phenotypes do not respond to antiepileptic drugs but are alleviated in most cases by giving large doses of pyridoxal 5′-phosphate (PLP). PLP is the active form of vitamin B6 participating in more than 100 enzymatic pathways. One of the causes of NEE is pathogenic mutations in the gene for human PNPO (hPNPO). PNPO is a key enzyme in converting pyridoxine (PN), the common dietary form of vitamin B6, and some other B6 vitamers to PLP. More than 25 different mutations in hPNPO, which result in reduced catalytic activity, have been described for PNPO-deficiency NEE. To date, no animal model is available to test new therapeutic strategies. In this report, we describe using zebrafish with reduced activity of Pnpo as an animal model. Knocking down zPnpo resulted in developmental anomalies including brain malformation and impaired locomotor activity, similar to the clinical features of PNPO-deficiency NEE. Other anomalies include a defective circulation system. These anomalies were significantly alleviated by co-injecting either zpnpo or hPNPO mRNAs. As expected from clinical observations in humans, supplementing with PLP improved the morphological and behavioral anomalies. PN only showed marginal positive effects, and only in a few anomalies. Remarkably, pyridoxamine (PM), another dietary form of vitamin B6, showed rescue effects even at a lower concentration than PLP, presenting a possible new therapeutic treatment for PNPO-deficiency NEE. Finally, GABA, a neurotransmitter whose biosynthesis depends on a PLP-dependent enzyme, showed some positive rescue effect. These results suggest zebrafish to be a promising PNPO-deficiency model for studying PLP homeostasis and drug therapy in vivo.
Highlights
The peptide sequence alignment and comparison show that zPnpo is 54% identical and 71% homologous to human PNPO (hPNPO) (Figures 2A, B)
As expected from the high sequence similarity between hPNPO and zPnpo (Figures 2A, D, E), the two structures predicted by the online program I-TASSER (Iterative Threading ASSEmbly Refinement) showed almost identical predicted folds that are close to the determined fold for hPNPO (Figure 2F) (Zhang, 2008; Roy et al, 2010; Yang et al, 2015)
Most patients only respond to the cofactor pyridoxal 5′-phosphate (PLP) as a treatment (Mills et al, 2005; Hoffmann et al, 2007; Ormazabal et al, 2008; Ghatge et al, 2012; Mills et al, 2014), while only a minor subset of patients respond to PN therapy (Mills et al, 2014; Plecko et al, 2014)
Summary
A severe neurological problem that presents in prenatal, neonatal, or infant children is neonatal epileptic encephalopathy (NEE) as a result of pyridoxine 5′-phosphate oxidase (PNPO) deficiency (Mills et al, 2005; Hoffmann et al, 2007; Ormazabal et al, 2008; Musayev et al, 2009; Balasubramaniam et al, 2010; Bowling, 2011; Ghatge et al, 2012; Mills et al, 2014; di Salvo et al, 2017a; di Salvo et al, 2017b; Wilson et al, 2019). Deficiency of PLP in cells is, likely to impact the development of many organs
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