Human pyridoxal 5'-phosphate oxidase (PNPO) is a key enzyme in vitamin B6 metabolism and is responsible for maintaining pyridoxal 5'-phosphate (PLP) homeostasis in tissues. PLP, the active form of vitamin B6, is a cofactor for over 160 known PLP-dependent enzymes serving vital roles in pathways involving sugars, lipids, amino acids, heme, and nucleotides. PLP-dependent enzymes participate in the metabolism of several neurotransmitters such as dopamine, serotonin, epinephrine, and gamma-aminobutyric acid. The activity of PLP-enzymes is essential for the correct functioning of the central nervous system, and relies on an appropriate availability of PLP in neuronal cells. Deficiency of PNPO, the enzyme that catalyzes the last step of PLP biosynthesis, results in a severe neonatal encephalopathy. Recent studies have widened the clinical phenotype of this condition and detected genetic variants of PNPO gene, whose pathogenetic role and clinical expression remain to be established.
The goal of the present project is the characterization of the functional effects of several newly discovered mutations in PNPO gene, in order to define their pathogenicity at a molecular level. Moreover, this project will investigate the transfer mechanism of PLP from PNPO to PLP-dependent enzymes responsible for neurotransmitter production. This is an often underestimated and never fully characterized feature, which could be related to the onset of epileptic phenotypes of patients showing PNPO mutations.
Our proposed studies also comprise patients screening, aimed at expanding the clinical framework currently associated with PNPO deficiency. We will evaluate the potential involvement of PNPO gene in the pathogenesis of a wider range of generalized idiopathic epilepsies, beyond the strictly neonatal age and showing different patterns of clinical severity. This would open up new therapeutic perspectives, extending the use of pyridoxine and PLP to other types of epileptic manifestations.
INNOVATION: Giving the importance of PLP homeostasis in the etiology of severe neurological diseases, we aim to clarify fundamental pathways concerning the conversion of apo-PLP-dependent enzymes into their catalytically active form (i.e., direct transfer of PLP to apo- PLP-dependent enzymes from PNPO) which in turn could be related to vitamin B6-dependent epileptic status of still uncharacterized patients carrying PNPO mutations. We will employ a wide range of interdisciplinary approaches (enzymology, structural biology, protein-protein interactions, and computational biology) to reveal how apo-PLP-dependent enzymes obtain PLP from wild type and mutant forms of PNPO. Vitamin B6 supports more vital bodily functions than any other vitamin, yet little information is currently available on the in vivo mechanism of how PLP, which must be kept at very low concentration in the cell to prevent toxic buildup, is transferred and distributed among competing apo-PLP-dependent enzymes. Inborn errors and inhibition of PNPO lead to PLP deficiency that is known or suspected to cause many pathologies, as mentioned above. In the future, more harmful polymorphisms in PNPO associated with disease states will be discovered. Understanding the role of vitamin B6 requires knowledge of its metabolism and regulation, especially regarding the functional role of its salvage pathway enzymes - among them, PNPO - and their importance in the biogenesis of PLP-dependent enzymes. In vivo studies alone cannot succeed until in vitro studies establish a mechanism for the conversion of apo-PLP-dependent enzymes to their active holo-forms. The first part of this proposal (AIM 1 and 2) describes in vitro studies establishing these mechanisms, that would be later tested through in vivo studies.
CLINICAL ASPECTS: Our proposed studies can potentially expand the clinical framework currently associated with PNPO deficiency (AIM 3). Demonstration of its role in the pathogenesis of epilepsy forms other than those described to date would open up new therapeutic perspectives, extending the use of pyridoxine and PLP to several types of epileptic manifestations. The availability of specifically-targeted treatments, especially if initiated early in those subjects presenting the onset of epileptic manifestations in the first 18 months of life, would allow to prevent multi-organ, multi-system complications, deficit in psychomotor development, and cognitive impairment; even in patients without significantly compromised psychomotor development this will undoubtedly improve the quality of life, reducing frequency, duration and severity of the clinical manifestations. Besides the overall improvement of the prognosis of the single affected patients, positive effects are expected in terms of reducing the cost for neurological follow-ups, emergency-related care, multi-organ complications and rehabilitative therapies. Additionally, the demonstration of the involvement of pathogenetic PNPO mutations in epileptic patterns other than those described so far would provide new data in the context of basic research to better understand the mechanisms of epileptogenesis and might allow to define more precisely the neurotransmitter networks and the mechanisms responsible for epileptogenesis.
FUTURE APPROACHES: In vivo studies have been envisaged as an interesting forthcoming goal of our project, which will be carried out in collaboration with our colleague Dr. Tzu-Fun Fu at the National Cheng Kung University, Taiwan. These studies are aimed at establishing the tropical freshwater fish zebrafish (Danio rerio) as inheritable epileptiform model for pathomechanistic study and drug discovery. Prof. Fu, who has a long-standing expertise on the manipulation of zebrafish, will produce PNPO knockout and knockdown lines of this organism. These would be employed to examine the effect of PNPO deficiency on development, through morphological and biochemical analyses, and test the outcome of B6 vitamers supplementation. Moreover, the rescue effect of the expression of wild type and mutant PNPO forms in knockout embryos will be tested by injecting PNPO coding sequences. Knocking down/out PNPO, the enzyme essential for generating active form of vitamin B6 in vivo, will impede embryonic development and give rise to the zebrafish displaying characteristics comparable to inheritable epilepsy. These in vivo studies on the effect of PNPO deficiency will be probably outside the time frame of the present proposal, but are integral part of a larger project which we plan to successfully carry out.
FEASIBILITY OF THE PROJECT: The longstanding experience of our research group on PNPO and the collaboration with external experienced and independently funded researchers will make our goal an actually achievable one. That is the reason why, although this research program may seem too ambitious - especially concerning the budget request - we feel confident to guarantee its success.