Enzymology of Pyrimidine Metabolism and ...

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Enzymology of Pyrimidine Metabolism and Neurodegeneration S. Vincenzetti, V. Polzonetti, D.Micozzi and S. Pucciarelli* School of Bioscience and Veterinary Medicine, University of Camerino, Camerino (MC), Italy Abstract: It is well known that disorders of pyrimidine pathways may lead to neurological, hematological, immunological diseases, renal impairments, and association with malignancies. Nucleotide homeostasis depends on the three stages of pyrimidine metabolism: de novo synthesis, catabolism and recycling of these metabolites.

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Cytidine and uridine, in addition to be used as substrates for pyrimidine nucleotide salvaging, also act as the precursors of cytidine triphosphate used in the biosynthetic pathway of both brain’s phosphatidylcholine and phosphatidylethanolamine via the Kennedy cycle. The synthesis in the brain of phosphatidylcholine and other membrane phosphatides can utilize, in addition to glucose, three compounds present in the blood stream: choline, uridine, and a polyunsaturated fatty acids like docosahexaenoic acid. Some authors, using rat models, found that oral administration of two phospholipid precursors such as uridine and omega-3 fatty acids, along with choline from the diet, can increase the amount of synaptic membrane generated by surviving striatal neurons in rats with induced Parkinson’s disease. Other authors found that in hypertensive rat fed with uridine and choline, cognitive deficit resulted improved. Uridine has also been recently considered as a neuroactive molecule, because of its involvement in important neurological functions by improving memory, sleep disorders, anti-epileptic effects, as well as neuronal plasticity. Cytidine and uridine are uptaken by the brain via specific receptors and successively salvaged to the corresponding nucleotides. The present review is devoted to the enzymology of pyrimidine pathways whose importance has attracted the attention of several researchers investigating on the mechanisms underlying the physiopathology of brain.

Keywords: Pyrimidine salvage, “de novo” pyrimidine, pyrimidine metabolism enzymes, pyrimidine homeostasis, nucleoside transport, brain phospholipids biosynthesis, neurological disorders. Received: December 10, 2015

Revised: February 28, 2016

1. INTRODUCTION Pyrimidine nucleotides have different important physiological roles inside the cell: first of all, they serve as precursors of nucleic acids, provide the activated UDP-sugars for protein glycosylation and glycogen synthesis and also provide the CDP-diacylglycerol phosphoglyceride for the assembly of cell membranes [1, 2]. An important role is played by uridine nucleotides since they regulate several physiological processes [3]. Furthermore, it is now well known that pyrimidines play an important role in the regulation of the central nervous system (CNS) and that metabolic

*Address correspondence to this author at the School of Bioscience and Veterinary Medicine, University of Camerino, Camerino (MC), Italy; E-mail: [email protected]

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Accepted: April 08, 2016

impairments affecting pyrimidine levels may lead to neurological disorders. Inside the cell, nucleotides can be synthesized by the “de novo pathways”, starting from simple metabolites, or recycled by the “salvage pathways”. The preference for one or the other route depends on the cell type, and on the stage of cell development itself. For example, the "de novo" pathway is actively present in cells in rapid proliferation, where the demand for nucleotides for DNA biosynthesis is rather high [4]. However, in some clinical situations or during development, nucleotides can be obtained also from the diet. Pyrimidine biosynthesis is up-regulated in cancer cells and the enzymes that catalyze the biochemical reactions of this pathway have been associated with the etiology or treatment of several diseases including AIDS, diabetes, and rheumatoid arthritis [5-7]. © 2016 Bentham Science Publishers

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2. PYRIMIDINE NUCLEOTIDES METABOLISM AND PHYSIOLOGICAL ROLE 2.1. Pyrimidines Uptake Pyrimidines are synthesized endogenously through the “de novo” and the “salvage” pathways, however there are conditions, such as disease, rapid growth or limited nutrient intake, where endogenous supply is not sufficient for normal body function. In these cases an exogenous supplement of these compounds through the diet may be essential to sustain growth and to maintain the cellular functions [8, 9]. In general dietary nucleotides are ingested mainly as nucleoproteins, which need to be enzymatically hydrolyzed before absorption (Fig. 1). First of all they are degraded by the proteases, the resulting nucleic acids are subsequently hydrolyzed in the stomach and then, thanks to pancreatic endonucleases and phosphodiesterases, are further degraded in the small intestine into nucleotides and finally to nucleosides thanks to an intestinal alkaline phosphatase (AP) and nucleotidases. The free bases are formed by the depletion of sugar moiety. Nucleotides have a reduced ability to cross cell membranes because of their negative charge and the absence of specific transporters, whereas nucleosides are able to enter in the epithelial cells thanks to facilitated diffusion and specific Na+-dependent carrier mediated mechanisms. More than 90% of dietary and endogenous nucleosides and bases are absorbed by the gut and are transported in the tissues by the circulating system [10]. In human and gerbil, uridine is the predominant circulating form, on the contrary in rats plasma the concentration of cytidine is higher with respect to uridine. From the enterocytes metabolic products of dietary and endogenous nucleotides enter the hepatic portal vein and are further metabolized in the hepatocytes. In the liver the nucleosides are salvaged to form the corresponding nucleoside triphosphates (NTP). At this level the “de novo” pathway is also active, which synthesizes nucleosides starting from simple precursor. From the liver pyrimidine nucleosides are finally released into the circulating system and are transported to the tissues. 2.2. Pyrimidine “De Novo” Biosynthetic Pathway and its Regulation Pyrimidine “de novo” biosynthetic pathway derives in part from the central metabolic precursors oxaloacetate and D-ribose 5-phosphate. L-aspartate, a precursor of pyrimidine ribonucleotides, is derived from oxaloacetate, which is generated in the TCA cycle.

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In Fig. (2) the reactions that lead to the formation of UMP are shown. Initially carbamoyl phosphate is synthesized from bicarbonate and ammonia in a multistep process, in which two molecules of ATP are hydrolyzed. The enzyme that catalyzes this reaction is carbamoyl phosphate synthetase II (1; CPS II, EC 6.3.5.5). This enzyme consists of two chains: one smaller chain which contains a site for glutamine hydrolysis for ammonia generation and a larger chain formed by two domains each of which catalyzes an ATP-dependent step. In the first step, bicarbonate is phosphorylated by ATP to form carboxyphosphate and ADP. Subsequently ammonia reacts with carboxyphosphate to form carbamic acid and inorganic phosphate. In the final step, carbamic acid is phosphorylated by another molecule of ATP to form carbamoyl phosphate. The product of the first reaction, carbamoyl phosphate reacts with aspartate to form carbamoylaspartate in a reaction catalyzed by aspartate transcarbamoylase (2; ATCase, EC 2.1.3.2). Carbamoylaspartate then cyclizes to form dihydroorotate in a reaction catalyzed by dihydroorotase (3; EC 3.5.2.3) which is then oxidized to orotate by the dihydroorotate dehydrogenase (4; DHODH, EC 1.3.3.1). Subsequently ribose, in the form of 5-phosphoribosyl-1-pyrophosphate (PRPP), is added to orotate to form oritidine 5’monophosphate (OMP). This reaction is catalyzed by orotate phosphoribosyltransferase (5; OPRT, EC 2.4. 2.10) and is driven by the hydrolysis of pyrophosphate. OMP is then decarboxylated to form uridine 5’monophosphate (UMP), in a reaction catalyzed by orotidine monophosphate decarboxylase (6; OMPD, EC 4.1.1.23). Once UMP is formed, nucleotide kinases convert UMP to UTP which serve as substrate for CTP synthetase (7; Ctps, EC 6.3.4.2) which catalyzes the formation of CTP with glutamine as the amino group donor. This pathway is universal, and is found in archaea, bacteria, fungi, plants and animals. The activities of CPS II, ATCase and dihydroorotase are part of a 240.0 kDa trifunctional protein named CAD, whereas the activities of OPRT and OMPD are on a bifunctional protein called UMP synthase (UMPS). CAD and UMPS are cytosolic and are localized around and outside the mitochondria, furthermore CAD seems to be associated with the cytoskeleton. The enzyme dihydroorotate dehydrogenase is a flavoprotein (FMN) with a molecular weight of 43.0 kDa and is localized in the inner mitochondrial membrane where it catalyzes the oxidation of dihydroorotate to orotate. Looking at the DHODH

Enzymology of Pyrimidine Metabolism and Neurodegeneration

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Fig. (1). Nucleotides absorption from the gut and in the bloodstream.

mechanism of action, dihydroorotate binds to a first binding site where is oxidized thanks to the FMN cofactor which is reduced; when orotate is released from the enzyme, FMNH2 is regenerated by the ubiquinone molecule. Therefore DHODH represents a functional link between the respiratory chain and the “de novo” pyrimidine biosynthesis [11]. Structural studies revealed that human DHODH has two domains: a catalytic domain in which is located the active site and small domain which forms a tunnel that leads to the active site and provides access to ubiquinone. This enzyme is inhibited by leflunomide, an immunosuppressive drug used in the treatment of rheumatoid arthritis, which binds within the tunnel, blocking access of ubiquinone to the active site [4]. The fact that mitochondria (where DHODH is located) are anchored to the cytoskeleton (where CAD is localized) facilitates a more efficient capture of dihydroorotate (produced by CAD) by DHODH and prevents the accumulation of carbamoyl aspartate in the cell [4]. The “de novo” pathway is regulated mainly at the level of CPS II that is subjected to a feedback inhibition by the end product UTP and is allosterically activated by phospho rybosyl pyrophosphate (PRPP), the substrate of UMPS. Furthermore PRPP contributes to the regulation of both pyrimidine and purine synthesis since it is also the substrate of the first step of purine biosynthesis. Several authors showed that the modulatory effect of UTP and PRPP may be modified by phosphorylation of CPS II at different sites: 1) by a cAMP-dependent protein kinase A (PKA) at level of S1406, abolishing UTP inhibition and decreasing the affinity of the enzyme for PRPP; 2) by a MAP kinase

(Erk1/2) cascade at level T456 converting UTP from an inhibitor to a modest activator and stimulating PRPP activation; 3) by an autophosphorylation at T1037 leading to an increased CPS II activity and modulation of the allosteric transitions [12-15]. The pool of UTP and CTP are regulated by CTP synthetase (Ctps) which is allosterically activated by GTP and is inhibited by the end product, CTP. Ctps activity is regulated by a phosphorylation process by both PKA and protein kinase C and, once phosphorylated, undergoes a tetramerization process after binding the substrates UTP and ATP that lead to its activation. In contrast, the dephosphorylated enzyme is not able to bind the substrates [16, 17]. The “de novo” synthesis of ribonucleotides is energetically very expensive, and organisms have adapted to salvage potential precursors from their environment. Since nucleotides, being negatively charged cannot cross the plasma membrane, salvage is limited to free bases and nucleosides. 2.3. Pyrimidine “Salvage Pathways” and its Regulation Pyrimidine bases are normally salvaged by a twostep way. Firstly, a pyrimidine nucleoside phosphorylase converts the pyrimidine bases to their respective nucleosides. The more specific nucleoside kinases then react with the nucleosides, forming monophosphate nucleotides. Further phosphorylation is carried out by increasingly more specific kinases (Fig. 3). Ribonucleotide reductase (9; RNR, EC 1.17.4.1) catalyzes the reduction of either ribonucleoside 5′-dior triphosphates (NDP or NTP) to corresponding deoxyribonucleoside 5′-di- or triphosphates [18]. There are three classes of RR that differ for the metal cofac-


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Fig. (2). “De novo” synthesis of pyrimidine nucleotides. The enzymes involved in the reactions leading to UMP formation are: (1) carbamoyl synthetase II (CPS II); (2) aspartate transcarbamoylase (ATCase); (3) dihydroorotase; (4) dihydroorotate dehydrogenase (DHODH); (5) orotate phosphoribosyltransferase (OPRT); (6) orotidine monophosphate decarboxylase (OMPD); (7) CTP synthetase (Ctps).

Fig. (3). “Salvage pathways” and degradation route of pyrimidine nucleotides. The enzymes involved are: (1) nucleoside phosphorylases/uridine phosphorylase (UP); (2) uridine-cytidine kinase (UCK); (3) UMP/CMP kinase; (4) nucleoside diphosphate kinase; (5) nucleoside phosphorylases/thymidine phosphorylase (TP); (6) thymidine kinase (TK); (7) cytidine deaminase (CDA); (8) deoxycytidine kinase (DCK); (9) ribonucleotide reductase (RNR); (10) Thymidylate synthase; (11) Dihydrofolate reductase (DHFR); (12) serine hydroxymethyltransferase (SHMT); (13) dihydropyrimidine dehydrogenase (DPD); (14) 5,6dihydrouracil dihydropyrimidinase (DHP); (15) β-ureidopropionase; (16) phosphopentomutase; (17) 5-phosphoribosyl-1pyrophosphate synthetase. R1P: Ribose-1-phosphate; R5P: Ribose-5-phosphate; PRPP: phosphoribosyl-1-pyrophosphate; FH2: dihydrofolate; FH4: tetrahydrofolate; m FH4: N5,N10-methylene tetrahydrofolate.


tors which serve to generate free radicals that lead to the formation of a thiyl radical (S•) necessary for the reduction mechanism. Uracil can be salvaged to form UMP through the concerted action of uridine phosphorylase (1; UP, EC 2.4.2.3) and uridine-cytidine kinase (2; UCK, EC:2.7.1.48): UP catalyzes the formation of uridine by adding ribose-1-phosphate (Rib-1-P) to uracil, whereas UCK phosphorylates this nucleoside into uridine monophosphate (UMP). UMP/CMP kinase (3; UMP/ CMPK EC:2.7.4.14) can phosphorylate UMP into uridine diphosphate, which is subsequently phosphorylated into UTP by a nucleoside diphosphate kinase. Analogously, thymidine phosphorylase (5; TP, EC 2.4.2.4) adds 2’deoxy-alpha-D-ribose 1-phosphate to thymine, forming deoxythymidine (TdR). Thymidine kinase (6; TK, EC 2.7.1.21) can then phosphorylate this compound into deoxythymidine monophosphate (dTMP) and subsequently into deoxythymidine diphosphate (dTDP) and deoxythymidine triphosphate (dTTP). The conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) is catalyzed by thymidylate synthetase (10; EC 2.1.1.45) by the following reaction (Fig. 3, inset a): N5,N10-methylene tetrahydrofolate + dUMP dihydrofolate + dTMP

By means of reductive methylation, deoxyuridine monophosphate (dUMP) and N5,N10-methylene tetrahydrofolate are together used to form dTMP, yielding dihydrofolate as a secondary product. N5,N10-methylene tetrahydrofolate is regenerated by two reactions: in the first one dihydrofolate reductase (11; DHFR, EC 1.5.1.3) reduces dihydrofolate to tetrahydrofolate by using NADPH as electron donor, whereas in the other one serine hydroxymethyltransferase (12; SHMT, EC 2.1.2.1) catalyzes the reversible, simultaneous conversion of L-serine to glycine and tetrahydrofolate to 5,10methylenetetrahydrofolate. The nucleosides cytidine and deoxycytidine can be salvaged along the uracil pathway by cytidine deaminase (7; CDA, EC 3.5.4.5), which converts them to uridine and deoxyuridine, respectively. UCK can phosphorylate cytidine into cytidine monophosphate (CMP), whereas deoxycytidine kinase (8; DCK, EC 2.7.1.74) catalyze the phosphorylation of deoxycytidine into deoxycytidine monophosphate (dCMP). UMP/ CMP kinase can phosphorylate (d)CMP into cytidine diphosphate or deoxycytidine diphosphate, which is phosphorylated into cytidine triphosphate or deoxy-


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cytidine triphosphate by a nucleoside diphosphate kinase. Intracellular nucleosides that derive either from dietary source or from endogenous nucleotides, if not recovered by the salvage pathways, are subjected to dephosphorylation by the specific nucleoside phosphorylases to form the corresponding free bases and the ribose/deoxyribose-1-phosphate. Pyrimidines are ultimately catabolized through reductive pathway in which the pyrimidine nucleotides are reduced to a β-amino acid, CO2 and ammonia. In particular uracil is firstly reduced to 5,6-dihydrouracil by a dihydropyrimidine dehydrogenase (13; DPD, EC 1.3.1.2), which is converted to β-ureidopropionate by 5,6-dihydrouracil dihydropyrimidinase (14; DHP, EC 3.5.2.2) and finally degraded to β-alanine, CO2 and NH3 by the action of the β-ureidopropionase (15; EC 3.5.1.6). Analogously thymine is converted to β-aminoisobutyrate, which can be further broken down into intermediates eventually leading into the citric acid cycle, CO2 and NH3. Ribose1-phosphate is converted to ribose-5-phosphate and subsequently to PRPP or is directed to the pentose phosphate pathway [19] (Fig. 3, inset b). In order to maintain an equilibrium between pyrimidine nucleosides and nucleotides pool, it is important the enzyme 5’-nucleotidase (5’-NT, EC 3.1.3.5) which catalyzes the dephosphorylation of 5’monophosphates to their corresponding nucleosides with the release of inorganic phosphate [20]. In addition to the hydrolase activity, 5’-NT shows a phosphotransferase activity between nucleotide donors and nucleoside acceptors. 5’-NT family comprises seven classes, among them one is extracellular, another one is GPI-anchored on the cell membrane surface and six are intracellular forms (one mitochondrial and five cytosolic). Two cytosolic 5’-NT enzymes, named cN-III and cdN are able to use pyrimidine nucleotides as substrate. In particular, cN-III is highly specific for the pyimidine base of the substrate and recognizes both oxy and deoxy sugar moiety. Regarding the phosphotransferase activity of cN-III, the enzyme is specific for (deoxy) cytidine and (deoxy) uridine as nucleoside acceptors and is also able to use some pyrimidine nucleoside analogs used as antitumor or antiviral drugs (AZT and AraC). Concerning the hydrolase activity the enzyme recognizes as physiological substrates CMP, UMP, dCMP, dUMP and several nucleoside 5’-monophosphates used in chemotherapy. cdN shows prevalently a hydrolase activity with preference for deoxynucleotides as substrates [20]. Both cytosolic cN-III and cdN are expressed in several mammalian tissues.


2.4. The Kennedy Pathway The pyrimidines uridine and cytidine also act as substrates for the biosynthesis of phosphatidylcholine (PC) and phosphatidyletanolamine (PE) through the Kennedy pathway [21]. The Kennedy pathway is divided in two branches: one based upon the formation of CDP-choline which leads to PC and another one based on the formation of CDP-ethanolamine that leads to PE (Fig. 4). In the first step of the PC pathway, choline is phosphorylated by an ATP-dependent reaction catalyzed by a choline kinase (CK), forming phosphocholine and ADP. Subsequently, a CTP:phosphocholine cytidylyltransferase (CCT) utilizes phosphocholine as substrate and CTP to form the high-energy intermediate cytidine diphosphocholine (CDP-choline) with the release of pyrophosphate. This second step is the ratelimiting step of the Kennedy pathway. In the third and last step, a CDP-choline: 1,2-diacylglycerol cholinephosphotransferase (CPT) uses CDP-choline and diacylglycerol (DAG) or alkyl-acylglycerol (AAG) as lipid anchor to form the final product phosphatidylcholine (PC) and CMP. The PE pathway is very similar to that described for the formation of PC, in this case the initial substrate is ethanolamine instead of choline. In this case the enzyme involved in the three steps of the reaction are respectively: ethanolamine kinase (EK),

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CTP:phosphoethanolamine cytidylyltransferase (ECT) and CDPethanolamine: 1,2-diacylglycerol ethanolaminephosphotransferase (EPT). Choline is a quaternary amine and is an essential nutrient grouped within the B-complex vitamins. It is mainly utilized for the synthesis of PC (by the Kennedy pathway), which in turn is a substrate for the synthesis of sphingomyelin by the sphingomyelin synthases, and it is a source of diacylglycerol, a second messenger signaling lipid. PC is a source of phosphatidic acid, lysophosphatidic acid, and arachidonic acid which is metabolized in other signalling molecules [22]. Choline is regenerated thanks to the hydrolysis of choline phospholipids by a phospholipase D that produces choline and phosphatidic acid, and by phospholipases A1 and A2 that generate free fatty acids and glycerolphosphocholine which is ultimately hydrolyzed to glycerol-3-phosphate and choline [22, 23]. Sphingomyelin through the action of a sphingomyleinases produces phosphocholine which can reenter in the Kennedy pathway. Animals must supply to the needs of ethanolamine mainly through the diet because, unlike the plants, they are not able to synthesize “de novo” this molecule; ethanolamine can be also obtained indirectly by pho-

Fig. (4). The Kennedy cycle and connections with pyrimidine metabolism. (1) Uridine-cytidine kinase (UCK); (2) UMP/CMP kinase; (3) nucleoside diphosphate kinase; (4) cytidine deaminase; (5) CTP synthetase; (6) choline kinase; (7) CTP:phosphocholine cytidyltransferase; (8) CDP-choline:1,2-diacylglycerol cholinephosphotransferase; (9) ethanolamine kinase; (10) CTP:phosphoethanolamine cytidyltransferase; (11) CDP-ethanolamine:1,2-diacylglycerol cholinephosphotransferase; (12) uridine phosphorylase (UP).


phatidylserine decarboxylation to phosphatidyletanolamine and subsequent release of ethanolamine by hydrolysis. Finally another way to obtain ethanolamine, even in small amount, is through the breakdown of sphingolipids by a sphingosine-1-phosphate lyase [23]. PE plays an important role in membrane architecture because of its particular tridimensional shape that introduces curvature stress in membranes influencing structure, folding, and activity of integral and peripheral membrane proteins. PE is also involved in cell division, it is a precursor of some biologically active molecules which can act as second messengers, it provides for the phosphoethanolamine capping of the glycosylphosphatidylinositol anchor which is required for attachment of proteins on the cell surface, and it is involved in posttranslational modifications. Several authors highlighted the involvement of PE in the inner membrane in mitochondria, suggesting that PE is important for the proper functioning of the respiratory chain and for the ubiquinone function [23]. 3. PYRIMIDINE TRANSPORT, METABOLISM AND PHYSIOLOGICAL ROLE IN BRAIN 3.1. Pyrimidine Transport in the Brain In humans the pyrimidine content of the brain is given by the uptake of uridine (predominantly) from the circulatory system [24], which is required for the maintenance of the brain electrophysiological activity. In other species such as rats the predominant nucleoside in plasma is cytidine, this difference is a consequence of the different species amount of cytidine deaminase which converts cytidine to uridine and therefore influences the principal pyrimidine in the blood (Fig. 3). In fact it has been observed that oral administration of cytidine (in the form of CDP-choline) elevates the plasma level of uridine in humans [24] while in rats lead to an increased cytidine level [25]. Nucleosides but also several nucleoside analogs with chemotherapeutic or antiviral activities can cross the plasma membranes thanks to specific nucleoside transporters (NT). NTs play a key role in nucleoside salvage pathways, in the functionality of nucleoside analogs prodrugs but also are indirectly involved in other physiological processes such as immune response, neurotransmission, coronary vasodilatation, and renal function [26]. There are two families of NTs: the SLC28 concentrative nucleoside transporters (CNT) and the SLC29 equilibrative nucleoside transporter (ENT) [27-29]. Table 1 shows a list of the nucleoside transporters with their properties, and substrate specificity. CNTs medi-


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ate the Na+ or Na+/H+-dependent accumulation of nucleosides in the cells and are found prevalently in intestinal and renal epithelia. CNTs are characterized by an high nucleoside affinity with a Km of 1-50 µM resembling the uridine and cytidine physiological plasma levels [30]. In humans three members of the CNT family with different affinities for the nucleosides are known: hCNT1 (selective for pyrimidine nucleosides), hCNT2 (selective for purine nucleosides and uridine), and hCNT3 which shows affinity for both purine and pyrimidine nucleosides. The differences in tissue distribution and in nucleoside specificity of hCNTs suggest that they possess different capacities in the nucleoside transport and probably different physiological and pharmacological roles. In line with this, there are some studies aimed to understand the permeant selectivities of hCNTs in order to develop nucleoside analogs prodrugs with good pharmacokinetic properties [31]. ENTs mediate the Na+-independent transport of nucleoside across the plasma membrane, are present in several types of cells [26] and are characterized by a low-affinity nucleoside transport with a Km of 100-800 µM [30]. Human ENTs protein family comprises four members hENT1, hENT2, hENT3, and hENT4. hENT1, hENT2 are selective for both purine and pyrimidine nucleosides. Also hENT3 possesses affinity for both purine and pyrimidine nucleosides but acts predominantly in the intracellular membranes. hENT4 has specificity for adenosine and functions mainly in the brain and in the heart [32, 33]. Furthermore several authors shown that some ENT members are able to transport purine and pyrimidine nucleosides and nucleobases with the same efficiency [28, 34] (Table 1). Cytidine and uridine from circulatory system can both be transported across the blood-brain barrier (BBB) by equilibrative transporters: particularly it has been shown that the transporters ENT1 and ENT2, cloned from rat and mouse BBB are able to transport cytidine and uridine across the BBB [35, 36]. Uridine may be transported also by the concentrative transporter CTN2, an evidence of its expression in the BBB was given by Li and coworkers [37] who have found that a recombinant CNT2 cloned from a rat BBB cDNA library behaved as a Na+-dependent transporter of adenosine and uridine. The CTN2 Km for uridine is very low, 9-40 µM therefore it may be able to mediate the transport of uridine across BBB under physiological conditions since plasma uridine level in human is about 3.1-4.9 µM [38]. Other authors instead suggested a localization for CTN2 at level of the endothelial membrane facing the brain’s interstitium [36]. Regarding cytidine, its concentration in human plasma is low


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Table 1. Nucleosides and nucleobases transporters (adapted from [54]). Type/Family

Characteristics

Type of transport

Proteins

Substrate(s)

Equilibrative/SLC29

- Low affinity for the substrate

Na+-independent

hENT1

Purine and pyrimidine nucleosides

hENT2

Purine and pyrimidine nucleosides, some nucleobase

hENT3

Purine and pyrimidine nucleosides and nucleobases

hENT4

Adenosine

hCNT1

Pyrimidine nucleosides and adenosine

hCNT2

Purine nucleosides and uridine

hCNT3

Purine and pyrimidine nucleosides & nucleoside analogues

facilitate diffusion

- Bidirectional

Concentrative/SLC28

- High affinity for the substrate

+

Na -dependent active transport

- Unidirectional

therefore it cannot satisfy the requirement of brain CTP because it is deaminated by cytidine deaminase which is present both in plasma and in cerebrospinal fluid. However cytidine is the principal circulating pyrimidine in rats but experimental data suggest that uridine concentration in brain rat’s extracellular fluids is seven-fold higher than cytidine and that uridine is effectively transported across the BBB [39]. It is known that cytidine can be transported only by the other two concentrative transporters CNT1 and CNT3 which have not yet been found in the BBB.

are involved in the glycogen synthesis or in the glycolysation processes. Furthermore UTP, once transformed into CTP by CTP synthetase, can contribute to the synthesis of brain phosphatidylcholine and phosphatidylethanolamine via the Kennedy pathway [21]. Therefore it may be supposed that there is a metabolic link between pyrimidine salvage and Kennedy pathway which could suggest the possible involvement of pyrimidines in some neurological disorders.

In the brain the “de novo” synthesis of both purine and pyrimidine nucleotides is very low whereas, as supported by several authors, in brain the salvage pathways, which are supplied with free bases and nucleosides by the bloodstream, are particularly active.

Structural analogues of nucleosides and nucleotides have been used in chemotherapy as potent anticancer and antiviral agents. These molecules act at the level of purine or pyrimidine metabolism by inhibiting the synthesis of nucleic acids; therefore several studies on nucleoside metabolism and related enzymes were performed and some other are still in progress in order to find more effective chemotherapeutic agents [40-42]. More recently, other studies have considered the possible involvement of nucleoside metabolism dysfunction in some neurological disorders, in fact a minimal change in a nucleoside concentration may affect several signal transduction pathways with the consequence of a modification of some neurochemical networks. Although the role of purine nucleosides, particularly adenosine and its receptors, in the mechanisms of neurodegeneration is well defined [43], the role of the pyrimidine nucleosides is not yet completely clear. Actually, cytidine and uridine are not known to have direct effects on CNS function. However, as discussed in the previous section, these two nucleosides, once salvaged into CTP and UTP contribute to the brain phosphatidylcholine and phosphatidylethanolamine synthesis through the Kennedy pathway suggesting that also uridine and cytidine may have a role in some neurode-

Uridine is the most present pyrimidine in the human brain since, as discussed previously, the CTN2 transporter has low affinity for this nucleoside and therefore can mediate the transport of uridine across BBB under physiological conditions. Once in the brain the homeostasis of uridine is maintained by the presence of UP, which is involved in the catabolism of uridine, and by the uridine-cytidine kinase (UCK) involved in the salvage of uridine to UTP and then to CTP (Fig. 4). Indeed, UCK regulates purine and pyrimidine salvage in the brain, in fact is inhibited by elevated concentrations of CTP and UTP, the final products of the salvage pathway, indications of sufficient amounts of pyrimidine nucleotides, and it is activated by ATP, signal of a sufficient amount of purines. Once formed, UTP can be used for the synthesis of dUTP, which is further processed to dTTP and is incorporated into DNA. UTP is also the precursor of the UDP-sugars (sugar conjugates such as UDP-glucose and UDP-galactose) which

3.2. Role of Pyrimidines in Brain Physiology


generative conditions. In particular, CTP is involved in the rate-limiting step of the Kennedy pathway, therefore it can be expected that the concentration of this nucleotide can influence the rate of membrane phosphatides synthesis. Several authors demonstrated that cytidine or uridine (as UMP) administration to rats or gerbils increased the level of neuronal cytidine and uridine which in turn increased the levels of CTP responsible for the increased synthesis of PC, and also led to an increased release of neurotransmitters (potassiuminduced striatal dopamine release) and neurite outgrowth [25, 44-48]. In some experiments it was shown that the oral administration of UMP to gerbils, resulted in an increased levels of pre- and post-synaptic proteins and neurite neurofibrillar proteins. If UMP is administered to adult gerbils together with the omega-3 fatty acid docosahexaenoic acid, there is a substantial increase of dendritic spines number together with an increase of membrane phosphatides and pre- and postsynaptic proteins in the hippocampus [49, 50]. Furthermore, pyrimidines can have effect on brain by the activation of purinergic receptors P2Y which belongs to the P2Y family of 7 transmembrane domain G-protein coupled receptors. Uridine derivatives recognizes the receptors P2Y2, P2Y4, P2Y6 which are localized in neurons and glial cells in several brain regions and the receptor P2Y14 which is localized in astrocytes and microglia. In humans the receptors P2Y4 and P2Y6, are activated by uridine nucleotides (UTP and UDP) [22-24] whereas UDP-glucose is responsible of the activation of P2Y14 receptors [51]. Other authors showed that UTP, UDP, CDP and CTP, through the P2Y2 receptors, are also able to modulate pain transmission [52, 53]. In a study conducted in PC12 rat pheochromocytoma cells, Pooler and coworkers [47] demonstrated that uridine mediated neurite outgrowth. In this experiment PC12 cells were differentiated by the nerve growth factor and were exposed to different uridine concentrations: after 4 days uridine promoted the increase of the number of neurites per cell in a dose-dependent manner. The neurite outgrowth was accompanied by the increase of phosphatidylinositol turnover which is, however, abolished by the P2Y receptor antagonists and by degradation of UTP by apyrase [47]. This indicate that uridine has a neurothropic effect by the stimulation of P2Y receptors mediated by UTP [38, 54]. Uridine seems also to modulate the release of the neurotransmitters dopamine and acetylcholine, in fact oral administration of UMP for a long period of time,


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caused an increased potassium-evoked dopamine release in the striatum and an elevated level of acetylcholine in striatum and striatal extracellular fluid [48, 55]. Furthermore it has been demonstrated in aged rats and in rats reared under impoverished environmental conditions, that CDP-choline supplemented by the diet is rapidly metabolized into choline and cytidine which is subsequently converted to uridine, which enters in the brain and is converted to UTP and CTP increasing the brain levels of membrane phosphatides, preventing hippocampal-dependent memory deficit but not striatal-dependent learning and memory. Similarly, in rats reared under impoverished environmental conditions and supplemented with UMP, instead of CDP-choline, the memory dysfunction resulted to be lightened [5658]. In another study conducted on normal adult gerbils the effect of uridine (in form of UMP, 0.5%), choline (0.1%) and docosahexaenoic acid (300 mg/kg/day), on the cognitive functions of these animals was examined. As a result it was found that the three compounds, orally administered, act in synergy increasing synaptic membrane content and dendritic spine formation and consequently enhance cognitive performances also in animals with normal cognitive function [59]. In this case the co-administration of UMP and docosahexaenoic acid increases the synthesis of brain PC: plasma uridine (which derived from UMP) can cross the BBB leading to an increase of brain uridine levels and consequently UTP, CTP and CDP-choline levels. CDPcholine may combine with docosahexaenoic acid to form PC, whereas UTP activates the PY2 receptors mediating neuronal differentiation and neurite outgrowth. Furthermore, the three compounds also act by enhancing the substrate-saturation of phosphatidesynthesizing enzymes [59]. These findings have important clinical implications for the treatment of neurodegenerative diseases in humans: in a study conducted on human older volunteers, it has been shown that dietary CDP-choline improved immediate and delayed logical memory, therefore it may be effective in treating age-related cognitive decline diseases such as Alzheimer's disease [60]. Subsequently, other studies demonstrated that CDP-choline is effective in the treatment of cognitive, emotional, and behavioral deficits associated with chronic cerebral disorders in the elderly [61] and that a long treatment with this molecule is well tolerated and effective since improves the post-stroke cognitive decline and enhances the functional recovery of patients through the enhancement of the endogenous mechanisms of neuro-


genesis and neurorepair [62]. Furthermore, in a randomized controlled study conducted on drug-naïve subjects with mild Alzheimer’s disease, it was shown that the administration of the specific nutrient combination Fortasyn Connect®, containing docosahexaenoic acid, UMP, choline and other nutrients (such as phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium) for 24 weeks can improve memory performance in mild Alzheimer’s disease patients most probably counteracting the progressive network disruption that occurs in Alzheimer’s disease [63, 64]. Finally, Saydoff and coworkers [65] observed that the oral administration of triacetyluridine (PN401), a bioavailable pro-drug of uridine, on a mice model of Huntington’s disease (HD) demonstrated neuroprotective effects; in fact all the primary features of HD in this model (mortality, neuronal degeneration, motor impairment) decreased after PN401 treatment. In addition to the involvement in brain lipid metabolism, uridine is also involved in carbohydrate metabolism, in particular in brain glycogen biosynthesis. The synthesis of glycogen requires energy that comes from UTP which reacts with glucose-1-phosphate to form UDP-glucose through a reaction catalyzed by the UTPglucose-1-phosphate uridylyltransferase. Glycogen synthase, which is expressed in astrocytes and neurons, catalyzes the glycogen chain elongation with the release of one molecule of UDP at each addition of a glucosyl residue. UDP, thanks to a nucleoside diphosphate kinase is then reconverted to UTP which can be used again for the synthesis of UDP-glucose. The role of endogenous sleep promoter substance (SPS) was also attributed to uridine, since this nucleoside was isolated in the brainstem of sleep-deprived rats [66]. Honda and coworkers [67] showed that the administration of uridine by intracerebroventricular infusion for 10h to rats, increased the frequencies of both slow wave sleep and paradoxical sleep but not their durations, whereas other authors, in an interesting work, demonstrated that localized electrolytic lesions made in the ventrolateral preoptic nucleus, a brain area described as a sleep center, eliminated the slow wave sleep-promoting effect of uridine [68, 69]. In another work by Sánchez and coworkers [70], it was found that UMP, although does not describe a clear circadian rhythm, increases its concentration during the period of darkness indicating a possible ultradian rhythm and a possible involvement of this nucleotide in the induction of a hypnotic mechanism. Besides the sleep-promoting

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effect, an anticonvulsant activity was also attributed to uridine and synergism with barbiturates was observed, suggesting a role of uridine on CNS depression including sedative and hypnotic activities [71]. 4. ENZYMOLOGY OF PYRIMIDINE METABOLISM AND NEUROLOGICAL FUNCTION From what has been described so far, it seems clear that the synthesis of UTP, and consequently the synthesis of CTP and phosphocholine depends on the availability of the pyrimidine precursors. In fact it has been shown that changes in the pyrimidine precursors concentration, in the kinetic properties of enzymes implied in pyrimidine metabolism may have a negative effect on the CNS. Errors in the nucleotide metabolism may cause a variation in the concentration of various substances in brain that may lead to neurodegeneration or developmental damage. Below are listed some enzymes of the pyrimidine metabolism, both of "de novo" and salvage pathways whose alterations are responsible of the different availability of the pyrimidine precursor and therefore could lead to pathological conditions. To facilitate the reading of the following subsections has been inserted a summary table that lists genes, enzymes and associated diseases and eventual therapeutic options for each disease (see Table 2). 4.1. Enzymes of the Pyrimidine “De Novo” Pathway As discussed in the section 2.2. the first 3 reactions of the “de novo” pyrimidine pathway are carried out by multifunctional enzymes CAD which comprises the activities of carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydroorotase. CPS II catalyzes the rate-limiting step in this pathway, is subjected to a feedback inhibition by UTP (the end product of the pathway) and is activated by PRPP. Huang et al., [72], performed studies on the signals involved in the regulation of CAD and observed a rapid decrease in CPS II activity during apoptosis, in fact they found two caspase-3 cleavage sites within the CSP II domain. In parallel with the loss of CAD activity a rapid decrease of pyrimidines in apoptotic cells was observed. The decrease in pyrimidine synthesis has negative consequences in phosphatidylcholine biosynthesis since this process is inactivated during apoptosis [73]. Recently some authors found a congenital disorder of glycosylation type 1Z (CDG1Z) caused by biallelic mutations in the CAD gene which causes an impaired aspartate incorporation into RNA and DNA through the



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Table 2. List of genes, related proteins of the pyrimidine “de novo” and salvage pathways, diseases associated to gene mutations and possible therapeutic options. Gene

ID

Location

Protein name

Pathway

Associated disease

Therapeutic options

CAD

790

2p22-p21

Trifunctional enzyme CAD

de novo

Congenital disorder of glycosylation 1Z (CDG1Z) [74]

Uridine administration

DHODH

1723

16q22

Dihydroorotate

de novo

Miller syndrome [78]

Not reported

Dehydrogenase UMPS

7372

3q13

Uridine 5’monophosphate synthase

de novo

Hereditary orotic aciduria [79]

Uridine administration

UPP1

7378

7p12.3

Uridine phosphorylase 1

salvage

Benign and malignant prostatic tissues [107]

Cancer therapy

TYMP

1890

22q13.33

Thymidine phosphorylase

salvage

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) [110-112]

Hemodialysis; Platelet infusions; Allogenig stem cell transplantation

TK2

7084

16q22-q23.1

Thymidine kinase-2 (TK2)

salvage

Mitochondrial DNA depletion syndrome (MDS) [119]

Dietary modulation; Cofactor supplementation; Liver transplantation; Stem cell transplantation

RRM2B

50484

8q23.1

p53-inducible ribonucleotide reductase (p53R2)

salvage

MDS 8A (Encephalomyopathic Type with Renal Tubulopathy); Autosomal dominant progressive external ophthalmoplegia-5; mitochondrial DNA Depletion Syndrome 8B (MNGIE Type) [147]

As described for TYMP and TK2 related diseases

DPYD

1806

1p22

Dihydropyrimidine dehydrogenase

salvage

Dihydropyrimidine dehydrogenase deficiency [153]

Not reported

DPYSD

1807

8q22.3

Dihydropyriminidase

salvage

Dihydropyriminidase deficiency [156]

Not reported

UPB1

51733

22Q11.23

β-Ureidopropionase

salvage

β-Ureidopropionase deficiency [161]

Not reported

de novo synthesis pathway and a decrease of CTP, UTP, and UDP-activated sugars that serve as donors for glycosylation. In this multisystem disorder a defect in glycoprotein biosynthesis occurs causing underglycosylated serum glycoproteins and resulting in several clinical features such as defects in the nervous system development, psychomotor retardation, dysmorphic features, hypotonia, coagulation disorders, and immunodeficiency. Uridine supplementation can reverse these symptoms therefore may be used as potential therapy for this glycosylation disorder [74]. The fourth step (reaction 4, Fig. 2) in the de novo biosynthesis of pyrimidine is catalyzed by the enzyme dihydroorotate dehydrogenase (DHODH). This enzyme is located in the inner membrane of mitochondria, whereas the other enzymes of the pyrimidine biosyn-

thesis are located in the cytosol. It has been shown by several authors that DHODH is functionally linked to the complex III of the respiratory chain, in fact the selective inhibition of complex III leads to a reduced DHODH activity and consequently to a reduced pyrimidine biosynthesis [11, 75-77]. Therefore a DHODH depletion can induce mitochondrial dysfunction, cell cycle arrest, and also an increased reactive oxygen species (ROS) production which leads to a decreased membrane potential and cell growth retardation [11]. Very recently, Khutornenko and coworkers [78] demonstrated that an impairment of DHODH function and, therefore of the of the “de novo” pyrimidine biosynthesis induces apoptosis in human colon cancer cells upon inhibition of the complex III of the respiratory chain.


Mutations in the DHODH gene cause Miller syndrome, a rare autosomal recessive disorder also known as postaxial acrofacial dysostosis. Clinically this disorder is characterized by distinctive craniofacial malformations such as micrognathia, cleft lip and/or palate, malar hypoplasia or aplasia of the postaxial elements of the limbs, coloboma of the eyelids. The mechanism through which DHODH mutations cause Miller syndrome is still unknown [79]. Uridine 5’-monophosphate synthase (UMPS, Fig. 2) is a bi-functional cytosolic enzyme which comprises the activities of orotate phosphoribosyltransferase, which is located in the N-terminal domain of the molecule and catalyzes the transfer from PRPP to orotate to form orotidine monophosphate (OMP) and orotidine monophosphate decarboxylase which is located in the C-terminal domain and converts OMP into UMP. An autosomal recessive disorder known as hereditary orotic aciduria, is caused by mutations in the UMPS gene, located on chromosome 3. Because of this disorder orotic acid is not converted to UMP and thus to other pyrimidines. UMP in fact is the precursor of UTP, CTP and TMP which act as feedback inhibitors of the initial reactions of the pyrimidine synthesis. In case of UMPS deficiency, there is a lack of CTP which maintains the enzyme aspartate transcarbamoylase (reaction 2, Fig. 2) efficient. This leads to an accumulation of plasma orotic acid to high concentrations, and is secreted in the urine (crystalluria). In this case, in homozygotes the urinary concentration of orotic acid can be of the order of mmoles/mmole creatinine whereas in healthy individuals is about one thousand fold less. Orotic aciduria is characterized by megaloblastic anemia insensitive to folic acid and vitamin B12 treatment, immunodeficiency and infections, whereas among neurological disorders, developmental delay, intellectual and motor impairment, hypotonia and strabysmus were observed. Megaloblastic anemia is due to the lack of CTP, TMP and UTP which leads to a decreased nucleic acid synthesis and decreased erythrocytes formation. Another consequence of UMPS impairment and therefore of the lack of CTP and UTP is the depletion of CDP-ethanolamine and CDP-choline (Fig. 4), necessary for phospholipids synthesis that should be considered as a cause of neurological damages. The type I orotic aciduria is characterized by a deficiency of both activities of UMPS, whereas the rarer type II is characterized by a deficiency of OMPD activity but elevated levels of OPRT [80]. However, in a more recent classification the orotic acid/orotidine (OA/OR) ratio is used to define the two types of orotic

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aciduria: in the type I there is a high OA/OR ratio whereas in type II this ratio is close to one [81]. Dietary administration of uridine leads to the remission of the orotic aciduria symptomatology. In fact dietary uridine, once passed the intestinal epithelium can reach the bloodstream, can easily cross the plasma membrane and inside the cell is converted to UMP by the enzyme uridine kinase and subsequently to UTP: in this way the “de novo” synthesis and the UMPS deficiency is bypassed. The formed UTP acts as inhibitor of carbamoyl phosphate synthetase II, leading to a decreased orotic acid synthesis. UTP is also the substrate of CTP synthetase, then exogenous uridine bypassing the defective UMPS, provides cells of UTP and CTP that are necessary for the synthesis of nucleic acids and other cellular function [82, 83]. The last step of the pyrimidine “de novo” biosynthesis is catalyzed by the rate-limiting enzyme CTP synthetase (Ctps) (Fig. 2). This enzyme catalyzes the ATP dependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. The final product CTP has a central role in the membrane phospholipids biosynthesis by the Kennedy and CDPdiacylglycerol pathways. Ctps has a glutaminase domain responsible of the glutamine deamination and a synthetase domain that aminates UTP (in a reaction ATP dependent) to form the product CTP. Ctps shows a binding site for the substrates (glutamine, UTP and ATP) and an allosteric site for GTP which acts as a positive allosteric activator of the glutaminase activity of the enzyme [84]. CTP acts as feedback inhibitor of Ctps and therefore regulates the membrane phospholipids biosynthesis. Ctps is active when is assembled in a tetrameric form and the availability of ATP, UTP, or CTP favors the tetramerization. In Saccaromyces cerevisiae two forms of Ctps have been identified and were named Ura7p and Ura8p showing a 78% identity in the aminoacid sequence and showing molecular masses of 64.7 and 64.5 kDa, respectively. The Ura7p enzyme is more abundant than the Ura8p form therefore is the most responsible of the CTP production in vivo [85, 86]. Furthermore it has been shown that the activities of both enzymes may be differently regulated in vivo by the cellular concentration of UTP and ATP [87]. Ctps enzymes (Ura7p and Ura8p) are allosterically inhibited by the product CTP and this regulation determines the intracellular concentration of CTP. The latter does not compete with UTP for the active site since it binds in two different sites [88].


The S. cerevisiae Ura7p form is phosphorylated by two different protein kinases (PKA, cyclic-AMP dependent and PKC, lipid-dependent). The phosphorylated enzyme showed an increased Vmax with respect to UTP and ATP and a decreased Km toward ATP, and the effect of CTP inhibition is less evident [89], furthermore it was demonstrated that the phosphorylation of Ctps allows the nucleotide-dependent tetramerization: a dephosphorylation of Ctps by alkaline phosphatese prevents the enzyme tetramerization and leads to a loss of enzymatic activity whereas a re-phosphorylation of the enzyme by kinases restores the teramer and the enzymatic activity [90]. Finally some authors have shown that different sites of phosphorylation are used by the two kinases and that the choice of the phosphorylation site affects the synthesis of lipids. In particular, phosphorylation at Ser36, Ser354, and Ser454 is correlated with an increase in phosphatidylcholine synthesis via the Kennedy pathway, instead the phosphorylation at Ser330 correlates with a decrease in the utilization of the Kennedy pathway [88]. From Chinese hamster ovary cells Ctps mutants defective in CTP product inhibition were isolated. These mutants showed to have resistance to the growth inhibitory effects of the chemotherapeutic agents 5fluorouracil and arabinosyl cytosine. The most frequent Ctps mutations were a glutamate-to-lysine and histidine-to-lysine mutation. It was shown that cells expressing the glutamate-to-lysine mutation have greater intracellular level of CTP and show alterations in the synthesis of membrane phospholipids when compared with cells that express the wild type forms of the enzyme [88, 91]. In particular, in these cells the synthesis of phosphatidylcholine, phosphatidylethanolamine, and phosphatidate was increased, while the synthesis of phosphatidylserine was decreased [88, 92]. The increased synthesis of phosphatidylcholine is due to an increased activity of CTP:phosphocholine cytidylyltransferase (CCT), the enzyme involved in the ratelimiting step of the CDP-choline branch of the Kennedy pathway. CCT activity is stimulated by high CTP levels: the cellular concentration of CTP is 0.7 mM and the apparent Km of CTP for the CCT is 1.4 mM, an increase of cellular CTP levels may affect positively the activity of this enzyme [88, 93]. Therefore an overexpression of Ctps activity leads to an increased cellular concentration of CDP-choline. Recent studies demonstrated that CTP synthetase is compartmentalized into filamentous structures called cytoophidia (meaning ‘cellular snakes’ in Greek) in the cytoplasm (Ccytoophidia) and in the nucleus (N-cytoophidia) of the eukaryotic cells. Both forms of Ctps, Ura7p and Ura8p,


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are able to form cytoophidia [94]. Until now it is not well known why a metabolic enzyme assembles into these snake-like sub-cellular structures, but surely they are an important aspect of the enzyme structure and function, in fact the ability to form filaments by CTP synthetase is highly conserved, since the cytoophidia were found in bacteria, yeast, Drosophila and human cells, indicating important physiological functions such as the regulation of the enzymatic activity [95-97]. Very recently, Barry and coworkers [98] found that Ctps polymerization is cooperative and inhibits the enzymatic activity, it is stimulated by CTP binding but is circumvented by the substrates UTP, ATP and glutamine. Therefore Ctps polymerization is not induced by the initial substrates but rather by the accumulation of the product CTP and once polymerized, Ctps is inactive and must disassociate to restore the original enzymatic activity. From a molecular point of view an explanation of the inactivity of the polymerized Ctps may be that the interactions between tetramers (responsible of the polymerization) sterically prevents a conformational change necessary for the Ctps activity [98]. In an interesting work, Aughey and coworkers [99] showed that cytoophidia formation in Drosophila tissues occurs during nutrient deprivation in cultured cells and also shown that cytoophidia are highly prevalent in the quiescent neuroblasts of the Drosophila larval central nervous system. Since Ctps activity is inhibited when the enzyme is incorporated into the filaments the authors speculated that a disassembly of cytoophidia is necessary to the nucleotides synthesis when these cells have a transition from a quiescent state to one of proliferation. Therefore the cytoophidia formation is reversible during neurogenesis thus indicating that the filament formation regulates the pyrimidine biosinthesis [99]. In conclusion, Ctps is a key enzyme in the pyrimidine de novo biosynthesis therefore is subjected to important regulatory strategies: it is regulated by multimer assembly, by allosteric modulators, end product inhibition, phosphorylation processes by protein kinases A and C, and recently was discovered that Ctps is also regulated by assembly into high ordered filaments (cytoophidia) for a finer control of its enzymatic activity. 4.2. Enzymes of the Pyrimidine Salvage Pathway With the aim of discussing about the enzymes involved in the pyrimidine salvage pathways and their involvement in neurodegenerative mechanisms, we will consider the central role of uracil and the enzymes that


lead to its formation and subsequent conversion UTP and the enzymes involved in the pathways that lead to its degradation to β-alanine (Fig. 3). Uridine Phosphorylase (UP, EC 2.4.2.3), is an important enzyme in the pyrimidine salvage pathway catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. UP is involved in the maintenance of uridine homeostasis together with thymidine phosphorylase (TP). UP is also able to activate pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. UP gene expression is strictly controlled at promoter level by oncogenes, tumor suppressor genes and cytokines; the enzymatic activity of UP is elevated in various tumor tissues and this induction makes the 5FU therapy more efficient for cancer patients [100, 101]. Some researchers studied the effect of UP inhibitors in order to increase the cellular level of uridine to counteract the toxic effect of fluoropyrimidines on healthy tissues during treatment [102]. In vertebrates two forms of UP were isolated: UP-1 and UP-2 which, in humans, shares about 62% identity. From phylogenetic analysis it is evident that the presence of two forms of UP arose early in vertebrates and are present in most mammals even if some of them have lost UP-2. Contrary to UP-2, UP-1 is overexpressed in tumor cells therefore could be interesting to develop a selective inhibitor capable of discriminate between the two UP homologous in order to raise the endogenous level of uridine which is involved in several physiological processes [103]. Uridine has a protective effect on astrocytes exposed to metabolic stress such as ischemia and UP seems to be involved in this effect. In fact Choi and coauthors [104, 105] investigated the effect of uridine on brain and neurons of in vivo and in vitro ischemic injury models in which it was found that orally administration of uridine reduced the number of ischemic episodes in the brain and that UP is highly expressed in the rat brain under this ischemic condition. Finally, Zhang and co-authors [106] reported that liver UP expression is regulated by some hepatic nuclear receptors involved in lipid metabolism, indicating a possible role of UP in hepatic lipogenesis or cholesterol transport. The same authors indicated the existence of a nuclear receptor signalling pathway that links lipid and uridine metabolisms. UP was reported to be component of the pathways leading to tumorigenesis and/or metastasis in various tumors. Very recently, it has been reported that UP-1 is typically expressed in benign and malignant prostatic tissues, therefore UP

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may have a role as therapeutic targets in prostate cancer [107]. Thymidine Phosphorylase (TP; EC 2.4.2.4) is a cytosolic enzyme of the pyrimidine salvage pathways catalyzing the reversible phosphorolysis of deoxythymidine and deoxyuridine to their respective bases and 2’-deoxy-D-ribose-1-phosphate. TP is expressed in several human tissues including brain, peripheral nerve, gastrointestinal system, bladder, lung and spleen [108]. TP is identical with an angiogenic factor, the “platelet-derived endothelial cell growth factor” (PD-ECGF) and is overexpressed in several solid tumors such as breast and colorectal cancer therefore is involved in angiogenesis, tumor growth and metastasis. The angiogenic effect and the chemotactic activity is given by 2-deoxy-D-ribose, the degradation product of thimidine which is generated by the enzyme [109]. A mutation on TP causes severe loss of the enzymatic activity and consequently an accumulation of deoxythymidine and deoxyuridine in the plasma and tissues. Since mitochondria rely on salvage pathways for the synthesis of their dNTP pool, the mitochondria assume the excess of deoxythymidine which is converted to deoxythymidine triphosphate (dTTP) by a mitochondrial thymidine kinase2. The excess of dTTP causes an imbalance of deoxynucleotide pools which can increase mutagenesis on the mitochondrial DNA (mtDNA). This alteration affects the proteins encoded by the mtDNA expecially those involved in the respiratory chain. The pathology involving thymidine phosphorylase is called “mitochondrial neurogastrointestinal encephalomyopathy” (MNGIE) [110, 111]. The symptoms of MNGIE are several, and consist in severe gastrointestinal dysmotility, extraocular muscle weakness, peripheral neuropathy, cerebral demyelination, impairment of the blood-brain barrier. The treatment of MNGIE consist in hemodialysis or platelet infusion, to reduce the deoxythymidine levels in blood, allogenic stem cell transplantation to restore TP activity (and consequently reduce deoxythymidine level in plasma) [112]. Thymidine Kinase (TK) is a deoxyribonucleoside kinase which catalyses the reaction of thymidine phosphorylation to TMP using ATP as phosphate donor. In mammalian cells, thyimidine kinase is present in two forms: thymidine kinase 1 (TK1, E.C. 2.7.1.21) which is present in the cytoplasm and is highly cell-cycle dependent, and thymidine kinase 2 (TK2, E.C. 2.7.1.21) which is located in the mitochondria and is cell-cycle independent. TK1 exists as homodimer or homotetramer with a subunit of about 25 kDA [113, 114].


TK1 level increases during the S phase of cell division, its level decreases during the G2/M phase and is degraded during mitosis. Human TK1 degradation occurs thanks to the ubiquitin-proteasome pathway [114-116]. Human TK1 is regulated at transcriptional, translational and post-translational level, furthermore is subjected to a feedback regulation by TTP, the final product of the reaction of thymidine phosphorylation. Recently, Chen et al. [117] found that TK1 play an important role in repair of DNA damage. High level of serum TK1 was found in several forms of cancer, therefore this enzyme is used as biomarker for detection of potential and early-stage tumors, for detection of cancer progression and antitumor therapy. Thymidine kinase 2 is the mitochondrial enzymatic form responsible of the phosphorylation of deoxythymidine, deoxycytidine and deoxyuridine to the corresponding 5’-monophosphate using ATP or CTP as phosphate donors. TK2 is subjected to a feedback inhibition by dTTP and dCTP. Rat mitochondrial TK2 is expressed mainly in brain, lung and spleen [118]. The dNTP mitochondrial pool is refurnished only by the salvage pathway therefore dysfunctions of the enzymes involved in this pathway can cause a mitochondrial DNA (mtDNA) depletion which results in organ dysfunction due to a reduced synthesis of respiratory chain component necessary for energy production. This leads to a genetically and clinically heterogeneous group of autosomal recessive disorders known as mtDNA depletion syndromes (MDS) [119]. MDS can affect a single organ or a combination of organs such as muscle, liver, brain, and kidney. Among the enzymes involved in the pyrimidine salvage pathways, it has been shown that point mutations, deletions or insertions on TK2 gene cause myopathic or hepatocerebral forms of MDS [120, 121]. Initially, TK2-related MDS were associated with myopathic forms: two mutations were described on TK2 gene which lead to a depletion of muscular mtDNA by Saada et al. [120], This myopathic form affects children before the age of 2 years and is characterized by hypotonia, generalized fatigue, decreased physical stamina, proximal muscle weakness, and feeding difficulty, however cognitive function is not impaired [119]. MDS are severe disorders with poor prognosis; no curative therapy is actually available for these disorders. Some therapeutic options for a number of MDS include dietary modulation, cofactor supplementation, liver transplantation, and stem cell transplantation [119].


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Recently it was shown that in humans, mutation on TK2 gene can cause neurological disorders thus indicating that TK2 seems to be important for neuronal functioning [122]. Furthermore other authors found that mice harboring a point mutation on TK2 (H126N) developed encephalomyelopathy with vacuolar changes in the spinal cord [123]. Bartesaghi and coworkers [124] focused the attention on the importance of TK2 in neuronal homeostasis. In particular, thanks to a TK2 knockout mouse model, they demonstrated that the loss of TK2 activity leads to a progressive depletion of mtDNA with consequent decreased amount of the electron transport chain protein in the brain. In TK2deficient neurons these anomalies cause impaired mitochondrial bioenergetic function, aberrant mitochondrial ultrastructure and neuronal degeneration. Finally, in a study on tissue distribution of TK2 it was shown that brain displayed a significantly higher activity of the enzyme than heart and liver [118]. In conclusion TK2 deficiency causes neuronal dysfunctions in vivo and indicates that brain, perhaps more than any other tissue, is dependent upon TK2 for the dNTP salvage. UMP/CMP Kinase (UMP-CMPK) catalyzes the transfer of a phosphate group from ATP to CMP, UMP, or dCMP, to form the corresponding diphosphate nucleotide. Curbo et al. [125] first demonstrated that in Drosophila melanogaster an UMP-CMP kinase is expressed in mitochondria. Subsequently, the full-length coding sequence of the human UMP-CMP kinase cDNA was cloned and it was found to be located in the mitochondria of HeLa cells: this novel protein was named UMPCMPK2 [126]. The mitochondrial enzyme showed kinetic properties significantly different from those of the cytosolic counterpart. In fact human cytosolic UMP-CMPK phosphorylated preferentially ribonucleotides, whereas mitochondrial UMP-CMPK2 showed higher efficacy for deoxyribonucleotides with dUMP as best substrate, followed by dCMP. The same authors showed that ATP acts as inhibitor of UMP-CMPK2 indicating that ATP could regulate the activity of the mitochondrial enzyme [126]. Therefore, in light of what has been said so far, UMPCMPK2 may be the main responsible for the synthesis of mitochondrial dUMP and dCMP and thus could play a role in the homeostasis of mitochondrial dNTP pool, as already seen for the mitochondrial enzyme TK2. It has been demonstrated that isolated brain mitochondria, the deoxypyrimidine phosphorylation activity


is very active and differs from the activities described in other tissue. In fact thymidine and deoxycytidine are transported across the inner membrane into the mitochondrial matrix which contains the enzyme necessary for their conversion into the corresponding triphosphates such as mitochondrial UMP-CMPK2, a TK2 with an increased expression, and a dTMP kinase [127]. Uridine-Cytidine Kinase (UCK; EC 2.7.1.48) is a rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway where it catalyzes the phosphorylation of the natural ribonucleosides cytidine and uridine to CMP and UMP, respectively. CMP and UMP are subsequently phosphorylated by mono- and diphosphate kinases to form the 5'-triphosphate nucleosides [128130]. UTP and CTP are potent feedback inhibitors of the kinase. Van Rompay et al. [131] found the presence of two members of UCK in human cells named UCK1(277 residues) and UCK2 (261 residues), both enzymes efficiently phosphorylate uridine and cytidine whereas no phosphorylation of adenosine or guanosine was detected. Although the two forms of UCK share about 70% sequence identity, the precise physiological role of UCK1 remains unknown, as UCK2 is responsible for the majority of uridine and cytidine phosphorylation in cells. Northern blot analyses performed to study the expression pattern of human UCK1 and UCK2 mRNAs revealed that UCK1was detected as two transcripts at high level of expression in liver, kidney, skeletal muscle, and heart, whereas low levels were present in brain, placenta, small intestine, and spleen. UCK2 was detected only in placentas [131], whereas an overexpression of UCK2 was found in rapidly dividing cells, including several tumor cell lines, and it was particularly high in the pancreatic tumor tissue [132]. Deoxycytidine Kinase (dCK; EC 2.7.1.74) is another rate-limiting enzyme in the salvage pathway with a broad substrate specificity. This enzyme catalyzes the 5′-phosphorylation of the natural substrates deoxycytidine, deoxyadenosine and deoxyguanosine to the corresponding 5’-monophosphates. The kinetics of the reaction is influenced by the nature of the phosphate group donor that can be either ATP or UTP [133]. The importance of dCK is due to the fact that in some mammalian cells it can contribute to the dTTP pools for the 75 %, in fact the dCK product dCMP is converted to dUMP by the action of dCMP deaminase and subsequently to dTTP trough the action of thymidylate synthase (see Fig. 2). dCK is expressed mainly in thy-

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mus, spleen, lymph nodes, stimulated blood mononuclear cells, and bone marrow cells, it shows an intermediate expression in lung, colon, placenta but its level is very low in differentiated tissues such as brain, liver, kidney, and muscle. dCK has also an important role in human therapy since it is able to activate several nucleoside analogs used in anticancer and antiviral therapy [133]. dCK is located mainly in the cytosol, as evinced by immunohistochemistry experiments with a dCK peptide antibody, however the enzyme shows also a nuclear localization signal sequence in the N-terminal region [134] but a nuclear localization of dCK was found only in dCK overexpressing cells. The product of the reaction catalyzed by dCK, dCMP, once phosphorylated to dCTP can be used, beside of DNA, also for the synthesis of “liponucleotides,” precursors of membrane phospholipids [135], however, it was demonstrated that in adult brain extracts there is a lack of dCK enzyme activity therefore the contribution to dCMP pool by dCK could be very low in the brain [136]. Cytidine Deaminase (CDA, EC 3.5.4.5) catalyzes the hydrolytic deamination of cytidine and deoxycytidine into uridine and deoxyuridine, respectively. This protein also deaminates several chemotherapeutic cytidine nucleoside analogs leading to their pharmacological inactivation. Human CDA is a homotetramer of about 60 kDa with a subunit size of 15 kDa [137]. Two nonsynonymous genetic variants, 79A>C (Lys27Gln) and 208G>A (Ala70Thr), have been found in the human CDA gene. Lys27Gln variant was found at 0.30-0.36 frequencies in Caucasians, at 0.20-0.21 in Japanese and at 0.04-0.10 in Africans. In contrast, the Ala70Thr variant was found at 0.13 in Africans and 0.04 in Japanese, but not in Caucasians and in African-Americans. These variants possess different kinetic properties which may affect the efficacy of cytidine nucleoside analogs uased in chemotherapy [138, 139] . CDA has been found and purified from different tissues: liver, normal and leukemic granulocytes, lymphoblasts and placenta [140-143]. The expression of CDA mRNA was observed at high levels in liver and placenta and at low levels in lung and kidneys. The presence of CDA mRNA was not detected in heart, brain and muscle, even if low activity of this enzyme found in these tissues by Ho [144]. The same author reported high CDA activity in liver and spleen and moderate activity in lung, kidney, large intestinal mucosa and colon mucosa.



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From above, it is demonstrated that in the brain dCK and CDA are present at very low levels; Ho [144] found that in human and mice brain the activity of CDA (114 and 142 nmoles/g/hr respectively) is at least 10 times higher than that of dCK (12 and 8 nmoles/g/hr respectively). This could mean that in brain the cytidine rather than being phosphorylated by kinases, is preferentially deaminated into uridine which is then phosphorylated to UMP and UTP, converted by the enzyme CTP synthetase to CTP which is also used for the biosynthesis of membrane phospholipids.

zymatic activity (activity site) and the other one controls the substrate specificity (substrate specificity site). ATP binds to the activity site and controls the overall activity switching “on” the enzymatic activity, while dATP, dGTP, or dTTP bind on the substrate specificity site and maintain balanced levels of all four deoxynucleotides. Because of its involvement in DNA biosynthesis, the level of RNR in mammalian cell is strictly related to the cell cycle and growth control mechanism, in fact the enzymatic activity reaches its maximum level during S-phase.

In support of this hypothesis, it was found that human CDA is inhibited by CMP with a Ki of 700 µM [137] and, more interestingly, by CTP with a Ki of 70 µM (unpublished results) which correspond to the physiological concentration of CTP found in the rat brain.

In humans, the RNR1 subunit is encoded by the RRM1 gene while there are two isoforms of the RNR2 subunit, encoded by the RRM2 and RRM2B genes. In particular, RRM2B encodes the small subunit of a p53inducible ribonucleotide reductase (p53R2). Unlike the S phase-specific RNR2 form, the p53R2 is induced in response to DNA damage and in proliferating cells by the p53 protein [146]. Mutations in RRM2B have been associated with mitochondrial DNA Depletion Syndrome 8A (Encephalomyopathic Type with Renal Tubulopathy), autosomal dominant progressive external ophthalmoplegia-5, and mitochondrial DNA Depletion Syndrome 8B (MNGIE Type) [147].

Ribonucleotide Reductase (RNR, EC) is the enzyme that catalyzes the conversion of the four 5’-di(or tri)-phospho-adenosine, -cytidine, -guanosine, and uridine to their 2’-deoxyribonucleotide counterparts. The importance of this enzyme lies in the fact that it provides the precursor for DNA biosynthesis or repair. One of the product of the enzymatic reaction, the 5’di(tri)phospho-2’-uridine is subsequently converted into thymidine by the enzyme thymidylate synthase before to be used for DNA biosynthesis [145]. Until now three main classes of RNR enzymes have been described depending on the metal cofactor utilized in the catalytic activity: diiron-oxygen cluster (class I); cobalt with a cobalamin cofactor (class II), iron-sulfur cluster coupled to S-adenosylmethionine (class III). All three classes have in the active site a cysteine residue that, thanks to the metal cofactor, is converted into a thiyl radical which, in turn, initiates the catalytic reaction. Class I RNR, has been found in all eukaryotic organisms and reduces ribonucleoside 5’-diphosphates to deoxyribonucleoside 5’-diphosphates, whereas class II and class II RNR are present in some bacteria and in some bacteriophages and reduce ribonucleoside 5’triphosphates to deoxyribonucleoside 5’-triphosphates. Class I RNRs are tetrameric enzymes (α2β2); the substrate binds to the active site located in the α2 homodimer constituted by the large subunit RNR1. The active site of RNR1 contains three conserved Cys residues and a glutamate residue. The other homodimer β2 is formed by the small subunit RNR2 which contains a tyrosyl radical deeply buried inside a hydrophobic environment, located close to an iron center that stabilize the tyrosyl radical [145]. In the subunit RNR1 there are two regulatory (allosteric) sites: one regulates the en-

RNR activity is inhibited by nitric oxide (NO) which is supplied by the NO-releasing molecules or by nitric oxide synthase activity. The inhibition is due to a nitrosation of the cysteines in the RNR1 subunit and to an inhibition of the tyrosyl radical in the RNR2 subunit [145]. It is well known that Reactive Oxygen Species (ROS), which also include NO, attack glial cells and neurons, leading to neuronal damage, an overproduction of free radicals can cause oxidative damage to biomolecules, (lipids, proteins, DNA) which may lead to several chronic diseases including neuronal disorders. For example the dopaminergic cell loss which causes Parkinson's disease (PD) is induced by NO through an inhibition of several enzymes involved in different pathways: cytochrome oxidase, mitochondrial complexes I, II, and IV in the respiratory chain, superoxide dismutase, glyceraldehyde-3-phosphate dehydrogenase; poly(ADP-ribose) synthase, lipid peroxidation, and protein oxidation, activation or initiation of DNA strand breakage, increased generation of toxic radicals such as hydroxyl radicals and peroxynitrite, and also ribonucleotide reductase [148]. An excess of NO may be cytotoxic either by combining with the above mentioned tyrosyl radical essential for the activity of RNR or by forming peroxynitrite which can generate, in some cases, free radicals [149].


Dihydropyrimidine dehydrogenase (DPD; EC 1.3.1.2) is the initial enzyme in the pyrimidine catabolic pathway where it catalyzes the rate-limiting reaction reducing uracil and thymine to the corresponding 5,6-dihydrouracil and 5,6- dihydrothymine, using NADPH as cofactor. This enzyme is expressed mainly in fibroblasts, liver, lymphocytes, monocytes, granulocytes, platelets but not in erythrocytes. Several studies demonstrated that this enzyme is an important regulatory enzyme in the metabolism of both the naturally occurring pyrimidines uracil and thymine as well as of some cancer chemotherapeutic drugs such as 5fluorouracil [150]. DPD deficiency is an autosomal recessive disease caused by mutations in the DPYD gene which frequently result in partial DPD deficiency in heterozygotes and in a severe deficiency in homozygotes. The heterozygous individuals, usually do not have related health problems but have an increased risk of complications if treated with some chemotherapeutic drugs [151]. In particular, in these cases the patients result asymptomatic until treated with a drug but present several symptoms when treated with 5-fluorouracil including gastroenteric disorders, myelosuppression and CNS alterations [152]. In homozygous individuals, the decreased or absent DPD activity leads to thymineuraciluria, the symptoms of DPD deficiency may include: developmental delays, mental retardation, hypertonia, microcephaly, delayed growth, autistic features and an evidence of severe delay in myelination of brain parenchyma [153]. Dihydropyrimidinase (DHP, EC 3.5.2.2), catalyzes the ring opening of 5,6-dihydrouracil and 5,6dihydrothymine to N-carbamyl-alanine and to Ncarbamyl-amino isobutyrate respectively (Fig. 3). DHP is an homotetrameric enzyme containing four tightly bound zinc ions per molecule of active enzyme [154], this enzyme is expressed mainly in liver and kidney. Three homologous cDNA clones were isolated from human fetal brain and termed “DHP related proteins” (DRPs) [155]. DHP deficiency is an autosomal recessive disorder characterized by elevated levels of the dihydropyrimidines in urine, blood, cerebrospinal fluid and by a variable clinical phenotype [156]. Since however, no DHP activity has been found in brain tissues, the presence of the dihydropyrimidines in cerebrospinal fluid may be due to a transport of these molecules across the blood-cerebrospinal fluid barrier [157]. DHP deficiency can cause neurological or gastrointestinal disorder in some individuals whereas in other individuals is asymptomatic. The neurological abnormalities

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are characterized by intellectual disability, seizures, and weak muscle tone (hypotonia). An abnormally small head size (microcephaly) and autistic behaviors that affect communication and social interaction also occur in some individuals with this condition. Gastrointestinal disorders in DHP deficiency include gastroesophageal reflux, cyclic vomiting, microvillous atrophy which cause nutrients malabsorption with consequent failure to thrive. Furthermore it has been shown that patients with DHP deficiency may incur in developing severe toxicity after administration of 5-fluorouracil, similarly to patients with DPD [158] deficiency. van Kuilenburg and coworkers [159] described the case of two siblings with a complete DHP deficiency and high levels of 5,6-dihydrouracil and 5,6-dihydrothymine in plasma, cerebrospinal fluid and urine. One of the two patients showed neurological complications whereas the other sibling was asymptomatic. The analysis of DHP gene (DPYS) revealed that both patients resulted heterozygous for two missense mutations, one in exon 6 (W360R) and another one in exon 7 (R412M). Since the residues W360 and R412 are not directly involved in the catalytic mechanism, because they are distant from the active site, their effect on the enzymatic activity is probably due to a global effect on protein folding and on the architecture of the enzyme. Recently, two novel single nucleotide polymorphisms (SNPs) in the exon 2 of DPYS have been detected in Japanese individuals: 285C > T (T95T) and 349T > C W117R which lead to a decreased DHP activity [160]. β-Ureidopropionase (EC 3.5.1.6) catalyzes the third step of the pyrimidine degradation pathway which is the conversion of N-carbamyl-β-alanine and Ncarbamyl-β-aminoisobutyric acid to β-alanine and βaminoisobutyric acid, ammonia and CO2 (Fig. 3). The importance of the pyrimidine degradation pathway lies in the fact that it is a source of β-alanine and βaminoisobutyric acid. β-alanine has a structural homology with γ-aminobutyric acid and glycine, the most important neurotransmitters in the CNS, and some authors suggested that β-alanine itself is involved in the synaptic transmission and in the regulation of the dopamine levels whereas the other product of the reaction, β-aminoisobutyric acid is a partial agonist of glycine receptor and it is involved in the stimulation of fatty acid oxidation and leptin secretion [161] . β-Ureidopropionase deficiency is characterized by strongly elevated levels of the N-carbamyl-ß-amino acids in urine and plasma in the affected individuals. The clinical manifestations are mainly neurological: MRI abnormalities, seizures, intellectual disabilities


and abnormal tonus regulation, and resulted more severe than those observed in patients with DPD and DPH deficiency [161]. From a pathogenic point of view, in patients with β-ureidopropionase deficiency the altered concentration of β-aminoisobutyric acid may affect the fatty acid oxidation and the leptin levels, thus contributing to the above mentioned neurological impairments. The human β-ureidopropionase gene (UPB1) has been mapped to chromosome 22q11.2 [162]. Subsequent mutations analysis of UPB1 in 16 patients with β-ureidopropionase deficiency, revealed 6 novel missense mutations and one splice-site mutation [161]. To investigate the effect of the 6 missense mutations on the activity of β-ureidopropionase, the mutant proteins were expressed in an E. coli strain with no endogenous enzymatic activity. The six mutants L13S, G235R, R236W, S264R, R326Q and T359M resulted completely inactive or with a significantly decreased βureidopropionase activity. In the mutants G235R, R236W and S264R the point mutations are located in the active site therefore they affect directly the substrate binding and the catalysis, whereas in the mutants L13S, R326Q and T359M the mutations affect the protein folding leading to structural instability. As in the case of the other two enzymes of the pyrimidine degradation DPD and DHP, also patients with β-ureidopropionase deficiency might develop 5-fluorouracil toxicity [163].


kind of molecules used as anti-neoplastic or antimicrobial drugs. Even though most of pyrimidines (as uridine) are non toxic as dietary components, and have access to the brain through transporters, it is still very complex to design a therapeutic approach for neurodegenerative diseases based on pyrimidines as lead compounds, due to lack of organ specificity and due to wide-spread side effects. Based on their action on central enzymes of nucleoside/nucleotide metabolism and neurotransmission, emerging pyrimidine compounds can be explored as promising therapeutic agents in CNS disorders. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS Declared none. REFERENCES [1] [2] [3] [4]

CONCLUSION Pyrimidine metabolism is involved in different metabolic functions: RNA, DNA and phospholipids synthesis; uridine, in the form of UDP sugars, is a cofactor in the enzymatic glycosylation reactions in the synthesis of glycogen, glycoproteins and glycolipids. From what we have seen so far, there is an important link between the phospholipids biosynthesis and metabolism of pyrimidine nucleotides; this implies that a change in the kinetic properties of the enzymes involved in the “de novo” or “salvage” pathways of pyrimidine metabolism may affect the availability of some pyrimidine precursors of phospholipids which, in turn, results in a negative effect on the central nervous system such as neurodegeneration or developmental damage. Due to the wide spectrum of physiological functions in which pyrimidines are involved it appears challenging, and at the same time compelling, to deeper elucidate the biochemical basis of the relationship between pyrimidine metabolism and neurotransmission. It is rather surprising in fact that only few nucleoside metabolism inhibitors have been developed for CNS applications, in comparison with the high number of this

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