Pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency in zebrafish results in fatal seizures and metabolic aberrations
Ciapaite, Jolita; Albersen, Monique; Savelberg, Sanne M C; Bosma, Marjolein; Tessadori, Federico; Gerrits, Johan; Lansu, Nico; Zwakenberg, Susan; Bakkers, Jeroen P W; Zwartkruis, Fried J T; van Haaften, Gijs; Jans, Judith J; Verhoeven-Duif, Nanda M
(2020) Biochimica et Biophysica Acta. Molecular Basis of Disease, volume 1866, issue 3
(Article)
Abstract
Pyridox(am)ine 5'-phosphate oxidase (PNPO) catalyzes oxidation of pyridoxine 5'-phosphate (PNP) and pyridoxamine 5'-phosphate (PMP) to pyridoxal 5'-phosphate (PLP), the active form of vitamin B6. PNPO deficiency results in neonatal/infantile seizures and neurodevelopmental delay. To gain insight into this disorder we generated Pnpo deficient (pnpo-/-) zebrafish (CRISPR/Cas9 gene editing). Locomotion analysis
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showed that pnpo-/- zebrafish develop seizures resulting in only 38% of pnpo-/- zebrafish surviving beyond 20 days post fertilization (dpf). The age of seizure onset varied and survival after the onset was brief. Biochemical profiling at 20 dpf revealed a reduction of PLP and pyridoxal (PL) and accumulation of PMP and pyridoxamine (PM). Amino acids involved in neurotransmission including glutamate, γ-aminobutyric acid (GABA) and glycine were decreased. Concentrations of several, mostly essential, amino acids were increased in pnpo-/- zebrafish suggesting impaired activity of PLP-dependent transaminases involved in their degradation. PLP treatment increased survival at 20 dpf and led to complete normalization of PLP, PL, glutamate, GABA and glycine. However, amino acid profiles only partially normalized and accumulation of PMP and PM persisted. Taken together, our data indicate that not only decreased PLP but also accumulation of PMP may play a role in the clinical phenotype of PNPO deficiency.
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Keywords: Pyridoxal 5′-phosphate (PLP), Pyridoxamine 5′-phosphate oxidase (PNPO) deficiency, Seizures, Vitamin B, Zebrafish, Molecular Medicine, Molecular Biology, Journal Article
ISSN: 0925-4439
Publisher: Elsevier
Note: Funding Information: This work was supported by a Wilhelmina Children's hospital Research fund grant to dr. M. Albersen. Funding Information: Here we present the first zebrafish model of PNPO deficiency generated using (CRISPR)/Cas9 gene editing technology, which reproduces both biochemical and clinical phenotypes of human disease, including the development of seizures. We show that PLP treatment is effective in normalizing PLP, glutamate, glycine and GABA content and in extending the survival of pnpo −/− zebrafish. Our data suggest that while PL(P) is essential in seizure control and survival, accumulation of PNPO substrates (PMP and/or PNP) may play a role in the clinical phenotype of PNPO deficiency and possibly contributes to the incomplete success of PLP treatment. Decreased availability of intracellular PLP is a common denominator in several genetic defects affecting human vitamin B 6 metabolism, including PNPO deficiency, pyridoxine-dependent epilepsy (caused by mutations in ALDH7A1 coding for α-aminoadipic semialdehyde dehydrogenase), and epilepsy caused by mutations in pyridoxal phosphate binding protein ( PLPBP ) and tissue-nonspecific alkaline phosphatase ( ALPL ), which all present with encephalopathy, often involving seizures [ 13 ]. For this reason, a low level of PLP in CSF is not a specific marker for PNPO deficiency [ 26 , 47 ]. In fact one PNPO-deficient patient with normal PLP in CSF has been reported [ 27 ]. PLP is essential in amino acid and neurotransmitter metabolism. Therefore, it is indispensable for normal brain development and function. We have shown previously that in CSF of healthy subjects, PLP and PL are the predominant B 6 vitamers, while PM is present only in low concentrations and PN is not detectable [ 38 , 48 , 49 ]. Decreased concentrations of PLP [ 2 , 10 , 26 , 28 ] and PL [ 2 ] in CSF of untreated PNPO deficient patients have been reported, with no information on the other B 6 vitamers. In agreement with these data, we show that PLP and PL were strongly decreased in pnpo −/− zebrafish at 20 dpf. The milder effect observed at 5 dpf could be explained by the fact that the yolk containing maternal vitamin B 6 is depleted at 5–6 dpf. Similar to the human situation, in which treatment of PNPO-deficient patients with PLP results in normalization of PLP in CSF [ 2 , 10 , 26 ], treatment of pnpo −/− zebrafish with PLP resulted in normalization of PLP and PL and in increased survival. Similarly, PNPO −/− HEK293 cells grown with PL in the culture medium had B 6 vitamer profiles comparable to WT cells, including normal PLP concentrations. It has been shown that PLP treatment can lead to strong elevations of PM in CSF [ 29 ] and that plasma PM concentrations are elevated in PNPO-deficient patients irrespective of vitamin B 6 treatment status [ 50 ]. We observed accumulation of PM in untreated and PLP-treated pnpo −/− zebrafish, and PNPO −/− HEK293 cells grown with PM in the culture medium, indicating that PM elevation is a feature of PNPO deficiency. In this study, PMP was as abundant as PLP at 5dpf and was the second most abundant B 6 vitamer (after PLP) at 20 dpf in WT zebrafish. PMP is almost not detectable in plasma of healthy humans [ 30 , 48 ] and rodents [ 16 , 51 ] and it is not detectable at all in CSF of healthy humans [ 38 , 48 , 49 ]. This is in agreement with the notion that PMP in body fluids should be rapidly dephosphorylated by ALPL in order to be taken up by tissue cells. Concentrations of PMP in healthy WT rodent tissues are as high or even higher than concentrations of PLP, depending on the type of tissue [ 16 , 51 ], suggesting that PMP measured in our zebrafish extracts is tissue-derived. Presumably, the bulk of intracellular PMP is generated during the catalytic cycle of transaminases, which comprise a large group of PLP-dependent enzymes involved in amino acid metabolism. The general transamination reaction proceeds in two steps: i) formation of an α-keto acid product and PMP (from PLP), followed by ii) formation of an amino acid product and regeneration of PLP (from PMP) [ 52 ]. The contribution of transaminases to tissue PMP is supported by the observation that PMP/PLP ratios are distinct in different rodent tissues and are hardly influenced by dietary vitamin B 6 [ 51 ]. We observed comparable PMP concentrations in WT HEK293 cells independent of whether they were supplied with PM, PN or PL in the culture medium. This further indicates that under WT conditions, PMP originates from the continuous activity of transaminases. Pnpo deficiency in zebrafish on the other hand resulted in strong accumulation of its substrates PMP and PNP, although quantitatively, PNP concentrations remained negligible. Much stronger accumulation of PMP could be explained by the fact that zebrafish feeds (from 16 dpf on only artemia) contained much more PM(P) than PN(P). Our data suggest that dietary B 6 vitamers may affect intracellular B 6 vitamer profiles in PNPO deficiency. B 6 vitamer profiles of PNPO −/− HEK293 cells cultured with PM, PN and PL support this notion. Unfortunately, the influence of dietary vitamin B 6 on the clinical phenotype of PNPO-deficient patients has never been investigated. Information on the effects of PNPO deficiency on vitamin B 6 metabolism in human tissues is available for the liver of a single PLP-treated PNPO deficient patient with cirrhosis, showing elevated content of PL and PA with no information on phosphorylated B 6 vitamers [ 53 ]. Therefore, our pnpo −/− zebrafish model provides the first insights in the effects of PNPO deficiency in animal tissues, showing that substrates of PNPO strongly accumulate and that this abnormality is not resolved by PLP treatment. The importance of accumulating PNPO substrates is supported by observations in plants, which utilize both de novo and salvage pathways of PLP synthesis. Mutations in pdx3 , the orthologue of human PNPO in plant Arabidopsis thaliana , result in accumulation of PMP, induction of stress-response pathways, abnormal amino acid metabolism and growth, with minimally affected PLP [ 54 ]. We show that in pnpo −/− zebrafish larvae, PLP treatment led to normalization of PLP but did not prevent PMP accumulation. This was accompanied by normalization of only glutamate and glycine, while several, mostly essential, amino acids remained elevated, suggesting a defect in their degradation. Catabolism of amino acids involve a transaminase (lysine and tryptophan require α-aminoadipate aminotransferase/kynurenine aminotransferase II, phenylalanine and tyrosine require tyrosine transaminase, arginine requires ornithine δ-aminotransferase, for example). A negative effect of high PMP concentrations on the activity of transaminases was already postulated in pdx3 mutants of A. thaliana [ 54 ]. However, a direct inhibitory effect of PMP on the activity of transaminases is not likely, because during the catalytic cycle of the enzyme, PLP-PMP-PLP transitions occur in the active site of the enzyme and transaminases do not bind PMP from the environment [ 52 ]. Our data from PNPO −/− HEK293 cells cultured with PM showed that while cells accumulated massive amounts of PMP, the total GOT activity (a representative transaminase) remained comparable to WT cells. The strong decrease observed in GOT activity in PNPO −/− HEK293 cells cultured with PN suggests that the activity of transaminases is largely dependent on cellular PLP, which was strongly decreased in these cells. Alternatively, failure of PLP treatment to normalize concentrations of the abovementioned essential amino acids in pnpo −/− zebrafish may be an indication of a more general tissue damage, possibly caused by PMP accumulation. Liver abnormalities, including hepatomegaly [ 2 ], abnormal liver function tests [ 31 , 53 , 55 ] and sometimes even hepatic cirrhosis [ 53 , 55 ], are common in PNPO-deficient patients, although it is not clear whether these abnormalities are an inherent feature of PNPO deficiency or whether they are caused by toxicity of PLP treatment itself. While in our study we cannot discriminate between different tissues due to the small zebrafish size, we could argue that pnpo −/− zebrafish display signs of defective liver metabolism based on for example elevated arginine and methionine concentrations, which are predominantly degraded in the liver. An intriguing observation was the completely normal PLP level in PNPO −/− HEK293 cells cultured in PM, in contrast to cells cultured in PN, where PLP was drastically reduced. This suggests that at least in some (human) cells an enzyme other than PNPO can lead to formation of PL(P) from PM(P). Hypothetically, at high concentrations PMP, which is a primary amine, could be oxidized by another oxidase than PNPO, resulting in formation of PL(P) as part of the PMP detoxification process. Candidate enzymes could be amine oxidases, for example monoamine oxidases with broad substrate specificity [ 56 ]. However, this requires further investigation. The fact that despite the abundance of PMP and PM in zebrafish feeds, PLP was decreased in pnpo −/− zebrafish, can be explained by relatively lower accumulation of PMP in pnpo −/− zebrafish compared to PNPO −/− HEK293 cells (10.8-fold and 35.8-fold, respectively). The validity of this notion can be tested by treating pnpo −/− zebrafish with high concentrations of PM(P). Similar to PNPO deficient humans and Drosophila melanogaster [ 57 ], pnpo −/− zebrafish developed severe seizures. The seizure onset was not neonatal, most likely due to the fact that some maternal vitamin B 6 was available to the mutant fish until the yolk was depleted around 5–6 dpf. We noted that the onset of seizures varied considerably among pnpo −/− siblings. This may be partially explained by the fact that zebrafish received a mix of feeds with varying vitamin B 6 composition and that some fish likely consumed more than others (based on variation of body weight). Seizures in pnpo −/− zebrafish led to death in a time window of a few hours. Treatment with PLP improved the survival rate drastically, presumably by abolishing the seizures through normalization of glutamate (excitatory neurotransmitter, and precursor of GABA), GABA and glycine (both inhibitory neurotransmitters). GABA has been previously implicated in seizures in zebrafish [ 44 ] and D. melanogaster [ 57 ]. Low concentration of glycine has been shown to exert pro-convulsive effect in drug-induced epilepsy in rats with attenuation of convulsions at higher glycine concentrations [ 58 ], showing the importance of glycine in regulating brain function. 5 Publisher Copyright: © 2019 The Author(s)
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