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  8. Molecular characterization of enzymes involved in vitamin B1- and B6-dependent epilepsies
Joint research project

Molecular characterization of enzymes involved in vitamin B1- and B6-dependent epilepsies

Project leaders
Angela Tramonti, Victoria Bunik
Agreement
RUSSIA - RFBR-suspended - Russian Foundation for Basic Research
Call
CNR/RFBR triennio 2018-2020 2018-2020
Department
Biomedical sciences
Thematic area
Biomedical sciences
Status of the project
New

Research proposal

Vitamins B1 and B6 play important roles in the metabolism of neurotransmitters such as glutamate, dopamine, serotonin, epinephrine, and gamma-aminobutyric acid [1]. The vitamin derivatives pyridoxal 5'-phosphate (PLP) and thiamine diphosphate (ThDP) are the cofactors of a plethora of enzymes, whose activity is of pivotal importance for central metabolism and is essential for the correct functioning of the central nervous system, which therefore relies on the appropriate availability of PLP and ThDP in neuronal cells [1, 2]. Deficiencies in the B1 and B6 vitamins, malfunctioning of enzymes involved in their metabolism, and impairment of enzymes using them as cofactors result in severe neurological disorders, such as vitamin-responsive epilepsies. Pharmacological treatment often employs B vitamins combined action, which may address possible interactions of their metabolism. In particular, the two collaborating groups have recently shown that increasing intracellular thiamine and ThDP may regulate a key enzyme of PLP metabolism, pyridoxal kinase (PLK) [3]. Remarkably, PLK is known to be a target of transcriptional factors with robust circadian rhythms, and deficiency in these transcriptional factors leads to generalized spontaneous and audiogenic epilepsies, associated with decreased brain levels of PLP, serotonin, and dopamine [4].
In particular, the focus of the project carried out by the Italian team regards vitamin B6-dependent epilepsies. PLP, the biologically active form of vitamin B6, acts as coenzyme in more than 160 distinct enzymatic activities involved in essential metabolic pathways. PLP-dependent decarboxylases participate to the metabolism of several biogenic amines acting as neurotransmitters such as dopamine, serotonin, epinephrine, norepinephrine, gamma-aminobutyric acid and histamine [5]. Mammals are not able to synthesize PLP as microorganisms and plants do, but they recycle it through a salvage pathway (in which PLK and pyridoxine 5'-phosphate oxidase are key components) from the different B6 vitamers contained in food and from protein turnover [6]. Once made available, PLP is somehow targeted to apo-PLP-enzymes. Indeed, deficiency in pyridoxine 5'-phosphate oxidase (PNPO), which is responsible for maintaining PLP homeostasis in tissues, results in severe epileptic encephalopathies [7]. In fact, the reduced activity of PNPO may have a profound effect on PLP levels in the brain, which may result in the unbalance of PLP-enzymes activities, with drastic consequences on brain function. Deficiency of vitamin B6 has been implicated in pathologies such as autism, schizophrenia, Alzheimer, Parkinson, epilepsy and Down's syndrome [2, 8]. Also PLK has been implicated in epileptogenesis and Parkinson [9, 10].
We will focus on a newly recognized and rare neurological disease known as neonatal epileptic encephalopathy (NEE), determined by autosomal recessive mutations in the gene encoding PNPO, that result in inadequate levels of PLP [11]. The main feature of the disease is the onset of severe seizures, often within hours from birth, which respond to PLP administration and, in some cases, also to pyridoxine. If not promptly treated, neonates will present a drug resistant epileptic status that might determine death. To date, 18 pathogenic mutations of the PNPO gene and about 40 patients have been reported in the literature [12-15]. In our laboratory, we have already characterized a missense mutation of human PNPO leading to NEE and found that it affected FMN binding, substrate binding and catalysis [16]. Recently, we have studied the functional effects of the new c.347G>A (p.R116Q) mutation of human PNPO gene, and discussed its pathogenetic role in epileptic encephalopathy [17]. The clinical manifestations observed in patients carrying this mutation expand the phenotype of PNPO deficiency to isolated mild epilepsy with later onset than previously reported, a peculiar clinical feature of these patients. Novel missense mutations have been found in children affected by NEE [11, 12, 18], but have yet not been characterized at molecular level. We believe that a detailed structural and functional characterization of these mutant forms is very important for a full understanding of the disease and for the rational devise of treatment strategies.
Moreover, regarding vitamin-responsive epilepsies, a novel ThDP-dependent 2-oxoacid dehydrogenase (DHTKD1) is particularly interesting, since the enzyme participates in catabolism of lysine and tryptophan, both associated with epilepsies [19].

1. Kennedy, 2016 Nutrients 8, 68.
2. di Salvo et al., 2012 Front Biosci (Elite Ed) 4, 897-913.
3. Mkrtchyan et al., 2015 Sci Rep 5, 12583.
4. Gachon et al., 2004 Genes Dev 18, 1397-412.
5. Clayton, 2006 Journal of Inherited Metabolic Disease 29, 317-26.
6. di Salvo et al., 2011 Biochimica Et Biophysica Acta-Proteins and Proteomics 1814, 1597-608.
7. di Salvo et al., 2011 Biochim Biophys Acta 1814, 1597-608.
8. Surtees et al., 2006 Future Medicine 1, 615-20.
9. Kang et al., 2002 Brain Res 957, 242-50.
10. Vilarino-Guell et al., 2010 Ann Neurol 67, 409-11.
11. Mills et al., 2005 Hum Mol Genet 14, 1077-86.
12. Mills et al., 2014 Brain 137, 1350-60.
13. Ruiz et al., 2008 Mol Genet Metab 93, 216-8.
14. Plecko et al., 2014 Neurology 82, 1425-33.
15. Jaeger et al., 2016 Mol Genet Metab Rep 6, 60-3.
16. Musayev et al., 2009 J Biol Chem 284, 30949-56.
17. di Salvo et al., 2017 Mol Genet Metab 122, 135-42.
18. Hoffmann et al., 2007 J Inherit Metab Dis 30, 96-9.
19. Stiles et al., 2016 JIMD Rep 25, 15-19.

Research goals

The goal of the present project is the molecular characterization of enzymes involved in vitamin B1 (Moscow) and B6 (Rome) metabolism, as potential targets of vitamin-responsive epilepsies. Our research unit will investigate the functional effect of several newly discovered mutations in PNPO gene, in order to define their pathogenicity at molecular level. Moreover, this project will consider the transfer mechanism of PLP from PNPO to PLP-dependent enzymes responsible for neurotransmitter production. Our proposed studies also comprise patients screening, aimed at expanding the clinical framework currently associated with specific genes, such as PNPO, but potentially including deficiencies of PLK and DHTKD1. 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. We will also characterize the role of DHTKD1 and PLK in epileptogenesis and perform studies on vitamin B1 and B6 homeostasis in epileptic rat lines. We also intend to set up enzymatic assays for diagnostic purposes to be used on samples such as tissue homogenates in animal models, fibroblast cultures and patients biological fluids.

Last update: 24/04/2024