Inborn Errors of Purine Salvage and Catabolism.

Cellular purine nucleotides derive mainly from de novo synthesis or nucleic acid turnover and, only marginally, from dietary intake. They are subjected to catabolism, eventually forming uric acid in humans, while bases and nucleosides may be converted back to nucleotides through the salvage pathways. Inborn errors of the purine salvage pathway and catabolism have been described by several researchers and are usually referred to as rare diseases. Since purine compounds play a fundamental role, it is not surprising that their dysmetabolism is accompanied by devastating symptoms. Nevertheless, some of these manifestations are unexpected and, so far, have no explanation or therapy. Herein, we describe several known inborn errors of purine metabolism, highlighting their unexplained pathological aspects. Our intent is to offer new points of view on this topic and suggest diagnostic tools that may possibly indicate to clinicians that the inborn errors of purine metabolism may not be very rare diseases after all.

The Good, the Bad and the New about Uric Acid in Cancer.

Uric acid is the final product of purine catabolism in man and apes. The serum concentration of uric acid is sex-, age- and diet-dependent and is maintained close to its maximal solubility, indicating that it plays some important role. Indeed, it has been demonstrated that, at physiological concentrations, uric acid is a powerful antioxidant, while at high intracellular concentrations, it is a pro-oxidant molecule. In this review, we describe the possible causes of uric acid accumulation or depletion and some of the metabolic and regulatory pathways it may impact. Particular attention has been given to fructose, which, because of the complex correlation between carbohydrate and nucleotide metabolism, causes uric acid accumulation. We also present recent results on the positive and negative effects played by uric acid in cancer and some new findings and hypotheses about the implication of this metabolite in a variety of signaling pathways, which can play a role in the pathogenesis of diseases such as metabolic syndrome, diabetes, and inflammation, thus favoring the development of cancer. The loss of uricase in Homo sapiens and great apes, although exposing these species to the potentially adverse effects of uric acid, appears to be associated with evolutionary advantages.

Cytosolic 5'-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway.

Cytosolic 5'-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.

Metabolic Aspects of Adenosine Functions in the Brain.

Adenosine, acting both through G-protein coupled adenosine receptors and intracellularly, plays a complex role in multiple physiological and pathophysiological processes by modulating neuronal plasticity, astrocytic activity, learning and memory, motor function, feeding, control of sleep and aging. Adenosine is involved in stroke, epilepsy and neurodegenerative pathologies. Extracellular concentration of adenosine in the brain is tightly regulated. Adenosine may be generated intracellularly in the central nervous system from degradation of AMP or from the hydrolysis of S-adenosyl homocysteine, and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. Inactivation of extracellular adenosine occurs by transport into neurons or neighboring cells, followed by either phosphorylation to AMP by adenosine kinase or deamination to inosine by adenosine deaminase. Modulation of the nucleoside transporters or of the enzymatic activities involved in the metabolism of adenosine, by affecting the levels of this nucleoside and the activity of adenosine receptors, could have a role in the onset or the development of central nervous system disorders, and can also be target of drugs for their treatment. In this review, we focus on the contribution of 5'-nucleotidases, adenosine kinase, adenosine deaminase, AMP deaminase, AMP-activated protein kinase and nucleoside transporters in epilepsy, cognition, and neurodegenerative diseases with a particular attention on amyotrophic lateral sclerosis and Huntington's disease. We include several examples of the involvement of components of the adenosine metabolism in learning and of the possible use of modulators of enzymes involved in adenosine metabolism or nucleoside transporters in the amelioration of cognition deficits.

Cytosolic 5'-Nucleotidase II Is a Sensor of Energy Charge and Oxidative Stress: A Possible Function as Metabolic Regulator.

Cytosolic 5'-nucleotidase II (NT5C2) is a highly regulated enzyme involved in the maintenance of intracellular purine and the pyrimidine compound pool. It dephosphorylates mainly IMP and GMP but is also active on AMP. This enzyme is highly expressed in tumors, and its activity correlates with a high rate of proliferation. In this paper, we show that the recombinant purified NT5C2, in the presence of a physiological concentration of the inhibitor inorganic phosphate, is very sensitive to changes in the adenylate energy charge, especially from 0.4 to 0.9. The enzyme appears to be very sensitive to pro-oxidant conditions; in this regard, the possible involvement of a disulphide bridge (C175-C547) was investigated by using a C547A mutant NT5C2. Two cultured cell models were used to further assess the sensitivity of the enzyme to oxidative stress conditions. NT5C2, differently from other enzyme activities, was inactivated and not rescued by dithiothreitol in a astrocytoma cell line (ADF) incubated with hydrogen peroxide. The incubation of a human lung carcinoma cell line (A549) with 2-deoxyglucose lowered the cell energy charge and impaired the interaction of NT5C2 with the ice protease-activating factor (IPAF), a protein involved in innate immunity and inflammation.

Purine-Metabolising Enzymes and Apoptosis in Cancer.

The enzymes of both de novo and salvage pathways for purine nucleotide synthesis are regulated to meet the demand of nucleic acid precursors during proliferation. Among them, the salvage pathway enzymes seem to play the key role in replenishing the purine pool in dividing and tumour cells that require a greater amount of nucleotides. An imbalance in the purine pools is fundamental not only for preventing cell proliferation, but also, in many cases, to promote apoptosis. It is known that tumour cells harbour several mutations that might lead to defective apoptosis-inducing pathways, and this is probably at the basis of the initial expansion of the population of neoplastic cells. Therefore, knowledge of the molecular mechanisms that lead to apoptosis of tumoural cells is key to predicting the possible success of a drug treatment and planning more effective and focused therapies. In this review, we describe how the modulation of enzymes involved in purine metabolism in tumour cells may affect the apoptotic programme. The enzymes discussed are: ectosolic and cytosolic 5'-nucleotidases, purine nucleoside phosphorylase, adenosine deaminase, hypoxanthine-guanine phosphoribosyltransferase, and inosine-5'-monophosphate dehydrogenase, as well as recently described enzymes particularly expressed in tumour cells, such as deoxynucleoside triphosphate triphosphohydrolase and 7,8-dihydro-8-oxoguanine triphosphatase.

Emerging Role of Purine Metabolizing Enzymes in Brain Function and Tumors.

The growing evidence of the involvement of purine compounds in signaling, of nucleotide imbalance in tumorigenesis, the discovery of purinosome and its regulation, cast new light on purine metabolism, indicating that well known biochemical pathways may still surprise. Adenosine deaminase is important not only to preserve functionality of immune system but also to ensure a correct development and function of central nervous system, probably because its activity regulates the extracellular concentration of adenosine and therefore its function in brain. A lot of work has been done on extracellular 5'-nucleotidase and its involvement in the purinergic signaling, but also intracellular nucleotidases, which regulate the purine nucleotide homeostasis, play unexpected roles, not only in tumorigenesis but also in brain function. Hypoxanthine guanine phosphoribosyl transferase (HPRT) appears to have a role in the purinosome formation and, therefore, in the regulation of purine synthesis rate during cell cycle with implications in brain development and tumors. The final product of purine catabolism, uric acid, also plays a recently highlighted novel role. In this review, we discuss the molecular mechanisms underlying the pathological manifestations of purine dysmetabolisms, focusing on the newly described/hypothesized roles of cytosolic 5'-nucleotidase II, adenosine kinase, adenosine deaminase, HPRT, and xanthine oxidase.

Cytosolic 5'-Nucleotidase II Silencing in a Human Lung Carcinoma Cell Line Opposes Cancer Phenotype with a Concomitant Increase in p53 Phosphorylation.

Purine homeostasis is maintained by a purine cycle in which the regulated member is a cytosolic 5'-nucleotidase II (cN-II) hydrolyzing IMP and GMP. Its expression is particularly high in proliferating cells, indeed high cN-II activity or expression in hematological malignancy has been associated to poor prognosis and chemoresistance. Therefore, a strong interest has grown in developing cN-II inhibitors, as potential drugs alone or in combination with other compounds. As a model to study the effect of cN-II inhibition we utilized a lung carcinoma cell line (A549) in which the enzyme was partially silenced and its low activity conformation was stabilized through incubation with 2-deoxyglucose. We measured nucleotide content, reduced glutathione, activities of enzymes involved in glycolysis and Krebs cycle, protein synthesis, mitochondrial function, cellular proliferation, migration and viability. Our results demonstrate that high cN-II expression is associated with a glycolytic, highly proliferating phenotype, while silencing causes a reduction of proliferation, protein synthesis and migration ability, and an increase of oxidative performances. Similar results were obtained in a human astrocytoma cell line. Moreover, we demonstrate that cN-II silencing is concomitant with p53 phosphorylation, suggesting a possible involvement of this pathway in mediating some of cN-II roles in cancer cell biology.

Interplay between adenylate metabolizing enzymes and AMP-activated protein kinase.

Purine nucleotides are involved in a variety of cellular functions, such as energy storage and transfer, and signalling, in addition to being the precursors of nucleic acids and cofactors of many biochemical reactions. They can be generated through two separate pathways, the de novo biosynthesis pathway and the salvage pathway. De novo purine biosynthesis leads to the formation of IMP, from which the adenylate and guanylate pools are generated by two additional steps. The salvage pathways utilize hypoxanthine, guanine and adenine to generate the corresponding mononucleotides. Despite several decades of research on the subject, new and surprising findings on purine metabolism are constantly being reported, and some aspects still need to be elucidated. Recently, purine biosynthesis has been linked to the metabolic pathways regulated by AMP-activated protein kinase (AMPK). AMPK is the master regulator of cellular energy homeostasis, and its activity depends on the AMP : ATP ratio. The cellular energy status and AMPK activation are connected by AMP, an allosteric activator of AMPK. Hence, an indirect strategy to affect AMPK activity would be to target the pathways that generate AMP in the cell. Herein, we report an up-to-date review of the interplay between AMPK and adenylate metabolizing enzymes. Some aspects of inborn errors of purine metabolism are also discussed.

The Inside Story of Adenosine.

Several physiological functions of adenosine (Ado) appear to be mediated by four G protein-coupled Ado receptors. Ado is produced extracellularly from the catabolism of the excreted ATP, or intracellularly from AMP, and then released through its transporter. High level of intracellular Ado occurs only at low energy charge, as an intermediate of ATP breakdown, leading to hypoxanthine production. AMP, the direct precursor of Ado, is now considered as an important stress signal inside cell triggering metabolic regulation through activation of a specific AMP-dependent protein kinase. Intracellular Ado produced from AMP by allosterically regulated nucleotidases can be regarded as a stress signal as well. To study the receptor-independent effects of Ado, several experimental approaches have been proposed, such as inhibition or silencing of key enzymes of Ado metabolism, knockdown of Ado receptors in animals, the use of antagonists, or cell treatment with deoxyadenosine, which is substrate of the enzymes acting on Ado, but is unable to interact with Ado receptors. In this way, it was demonstrated that, among other functions, intracellular Ado modulates angiogenesis by regulating promoter methylation, induces hypothermia, promotes apoptosis in sympathetic neurons, and, in the case of oxygen and glucose deprivation, exerts a cytoprotective effect by replenishing the ATP pool.

Mitochondrial Damage and Apoptosis Induced by Adenosine Deaminase Inhibition and Deoxyadenosine in Human Neuroblastoma Cell Lines.

The treatment with deoxycoformycin, a strong adenosine deaminase inhibitor, in combination with deoxyadenosine, causes apoptotic cell death of two human neuroblastoma cell lines, SH-SY5Y and LAN5. Herein we demonstrate that, in SH-SY5Y cells, this combination rapidly decreases mitochondrial reactive oxygen species and, in parallel, increases mitochondrial mass, while, later, induces nuclear fragmentation, and activation of caspase-8, -9, and -3. In previous papers we have shown that a human astrocytoma cell line, subjected to the same treatment, undergoes apoptotic death as well. Therefore, both astrocytoma and neuroblastoma cell lines undergo apoptotic death following the combined treatment with deoxycoformycin and deoxyadenosine, but several differences have been found in the mode of action, possibly reflecting a different functional and metabolic profile of the two cell lines. Overall this work indicates that the neuroblastoma cell lines, like the line of astrocytic origin, are very sensitive to purine metabolism perturbation thus suggesting new therapeutic approaches to nervous system tumors. J. Cell. Biochem. 117: 1671-1679, 2016. © 2015 Wiley Periodicals, Inc.

IMP-GMP specific cytosolic 5'-nucleotidase regulates nucleotide pool and prodrug metabolism.

BACKGROUND: Type II cytosolic 5'-nucleotidase (cN-II) catalyzes the hydrolysis of purine and, to some extent, of pyrimidine monophosphates. Recently, a number of papers demonstrated the involvement of cN-II in the mechanisms of resistance to antitumor drugs such as cytarabine, gemcitabine and fludarabine. Furthermore, cN-II is involved in drug resistance in patients affected by hematological malignancies influencing the clinical outcome. Although the implication of cN-II expression and/or activity appears to be correlated with drug resistance and poor prognosis, the molecular mechanism by which cN-II mediates drug resistance is still unknown. METHODS: HEK 293 cells carrying an expression vector coding for cN-II linked to green fluorescent protein (GFP) and a control vector without cN-II were utilized. A highly sensitive capillary electrophoresis method was applied for nucleotide pool determination and cytotoxicity exerted by drugs was determined with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. RESULTS: Over-expression of cN-II causes a drop of nucleoside triphosphate concentration and a general disturbance of nucleotide pool. Over-expressing cells were resistant to fludarabine, gemcitabine and cytarabine independently of cN-II ability to hydrolyze their monophosphates. CONCLUSIONS: An increase of cN-II expression is sufficient to cause both a general disturbance of nucleotide pool and an increase of half maximal inhibitory concentration (IC50) of the drugs. Since the monophosphates of cytarabine and gemcitabine are not substrates of cN-II, the protection observed cannot be directly ascribed to drug inactivation. GENERAL SIGNIFICANCE: Our results indicate that cN-II exerts a relevant role in nucleotide and drug metabolism through not only enzyme activity but also a mechanism involving a protein-protein interaction, thus playing a general regulatory role in cell survival. SENTENCE: Resistance to fludarabine, gemcitabine and cytarabine can be determined by an increase of cN-II both through dephosphorylation of active drugs and perturbation of nucleotide pool.

Cytosolic 5'-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf.

IMP/GMP preferring cytosolic 5'-nucleotidase II (cN-II) is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR) domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain) was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.

The combination of adenosine deaminase inhibition and deoxyadenosine induces apoptosis in a human astrocytoma cell line.

Alterations in the functions of astrocytes contribute to the appearance of a variety of neurological pathologies. Gliomas, especially those of astrocytic origin, are particularly resistant to chemotherapy and are often characterized by a poor prognosis. Neuroblastoma is the tumour with the higher incidence in infants. Anticancer drugs can induce apoptosis and their cytotoxic effect is often mediated by this process. We have previously demonstrated that the combination of deoxycoformycin, a strong adenosine deaminase inhibitor, and deoxyadenosine is toxic for a human astrocytoma cell line. In fact, after 15 h of treatment, this combination increases both mitochondrial reactive oxygen species and mitochondrial mass, induces apoptosis as indicated by cytochrome c release from mitochondria and activation of caspase-3. These events are preceded by reduction in lactate release in the medium. In this work we demonstrate that after 8 h of incubation with deoxyadenosine and deoxycoformycin, caspase-8 is activated, mitochondrial mass increases and mitochondrial reactive oxygen species decrease. The addition of baicalein to the incubation medium reduces cell death and caspase-3 activity induced by deoxycoformycin and deoxyadenosine in combination. This protective effect is correlated to an increase of lactate released in the medium, a decrease in the intracellular levels of dATP, and an increase in ATP levels, as compared with the cells subjected to the treatment with deoxycoformycin and deoxyadenosine without any further addition. The effect of baicalein appears to be related to an inhibition of deoxyadenosine phosphorylation, rather than or in addition to the well known antioxidant activity of the compound. This work indicates that an astrocytoma cell line, reported to be resistant to mitochondria-dependent pathways of apoptosis, is indeed very sensitive to a manipulation affecting the balance of cellular purine metabolite concentrations. The same treatment is also cytotoxic on a neuroblastoma cell line, thus suggesting long term implications for cancer therapy.

A native electrophoretic technique to study oligomerization and activity of cytosolic 5'-nucleotidase II.

The analysis of the oligomeric active state of a native protein usually requires the application of at least two analytical methods such as gel filtration and analytical ultracentrifugation. Both methods require a substantial amount of protein, time and/or expensive equipment. We here describe a native electrophoretic method for the identification of the native molecular weight of the recombinant wild-type cytosolic 5'-nucleotidase (cN-II) and of its mutants in subunit interfaces Y115A, F36R, K311A and G319Q. The protein was stained both with protein dye and with an activity staining method. Our results demonstrated that purified recombinant protein preparations contained substantial amounts of nucleic acids and misfolded, inactive protein. Furthermore, cN-II mutants K311A and G319Q in subunit interface assume a quaternary dimeric active form, while the only active quaternary structure of wild-type cN-II is the tetramer.

Expression of bovine cytosolic 5'-nucleotidase (cN-II) in yeast: nucleotide pools disturbance and its consequences on growth and homologous recombination.

Cytosolic 5'-nucleotidase II is a widespread IMP hydrolyzing enzyme, essential for cell vitality, whose role in nucleotide metabolism and cell function is still to be exactly determined. Cytosolic 5'-nucleotidase overexpression and silencing have both been demonstrated to be toxic for mammalian cultured cells. In order to ascertain the effect of enzyme expression on a well-known eukaryote simple model, we expressed cytosolic 5'-nucleotidase II in Saccharomyces cerevisiae, which normally hydrolyzes IMP through the action of a nucleotidase with distinct functional and structural features. Heterologous expression was successful. The yeast cells harbouring cytosolic 5'-nucleotidase II displayed a shorter duplication time and a significant modification of purine and pyrimidine derivatives concentration as compared with the control strain. Furthermore the capacity of homologous recombination in the presence of mutagenic compounds of yeast expressing cytosolic 5'-nucleotidase II was markedly impaired.

Novel metabolic aspects related to adenosine deaminase inhibition in a human astrocytoma cell line.

Adenosine deaminase, which catalyzes the deamination of adenosine and deoxyadenosine, plays a central role in purine metabolism. Indeed, its deficiency is associated with severe immunodeficiency and abnormalities in the functioning of many organs, including nervous system. We have mimicked an adenosine deaminase-deficient situation by incubating a human astrocytoma cell line in the presence of deoxycoformycin, a strong adenosine deaminase inhibitor, and deoxyadenosine, which accumulates in vivo when the enzyme is deficient, and have monitored the effect of the combination on cell viability, mitochondrial functions, and other metabolic features. Astrocytomas are the most common neoplastic transformations occurring in glial cell types, often characterized by a poor prognosis. Our experimental approach may provide evidence both for the response to a treatment affecting purine metabolism of a tumor reported to be particularly resistant to chemotherapeutic approaches and for the understanding of the molecular basis of neurological manifestations related to errors in purine metabolism. Cells incubated in the presence of the combination, but not those incubated with deoxyadenosine or deoxycoformycin alone, underwent apoptotic death, which appears to proceed through a mitochondrial pathway, since release of cytochrome c has been observed. The inhibition of adenosine deaminase increases both mitochondrial reactive oxygen species level and mitochondrial mass. A surprising effect of the combination is the significant reduction in lactate production, suggestive of a reduced glycolytic capacity, not ascribable to alterations in NAD⁺/NADH ratio, nor to a consumption of inorganic phosphate. This is a hitherto unknown effect presenting early during the incubation with deoxyadenosine and deoxycoformycin, which precedes their effect on cell viability.

Initial studies to define the physiologic role of cN-II.

IMP preferring cytosolic 5'-nucleotidase II (cN-II) is a widespread enzyme whose amino acid sequence is highly conserved among vertebrates. Fluctuations of its activity have been reported in some pathological conditions and its mRNA levels have been proposed as a prognostic factor for poor outcome in patients with adult acute myeloid leukemia. As a member of the oxypurine cycle, cN-II is involved in the regulation of intracellular concentration of 5'-inosine monophosphate (IMP), 5'-guanosine monophosphate (GMP), and also 5-phosphoribose 1-pyrophosphate (PRPP) and is therefore involved in the regulation of purine and pyrimidine de novo and salvage synthesis. In addition, several studies demonstrated the involvement of cN-II in pro-drug metabolism. Notwithstanding some publications indicating that cN-II is essential for the survival of several cell types, its role in cell metabolism remains uncertain. To address this issue, we built two eucaryotic cellular models characterized by different cN-II expression levels: a constitutive cN-II knockdown in the astrocytoma cell line (ADF) by short hairpin RNA (shRNA) strategy and a cN-II expression in the diploid strain RS112 of Saccharomyces cerevisiae. Preliminary results suggest that cN-II is essential for cell viability, probably because it is directly involved in the regulation of nucleotide pools. These two experimental approaches could be very useful for the design of a personalized chemotherapy.

Metabolic network of nucleosides in the brain.

Brain relies on circulating nucleosides, mainly synthesised de novo in the liver, for the synthesis of nucleotides, RNA, nuclear and mitochondrial DNA, coenzymes, and pyrimidine sugar- and lipid-conjugates. Essentially, the paths of nucleoside salvage in the brain include a two step conversion of inosine and guanosine to IMP and GMP, respectively, and a one step conversion of adenosine, uridine, and cytidine, to AMP, UMP, and CMP, respectively. With the exception of IMP, the other four nucleoside monophosphates are converted to their respective triphosphates via two successive phosphorylation steps. Brain ribonucleotide reductase converts nucleoside diphosphates to their deoxy counterparts. The delicate qualitative and quantitative balance of intracellular brain nucleoside triphosphates is maintained by the relative concentrations of circulating nucleosides, the specificity and the K(m) values of the transport systems and of cytosolic and mitochondrial nucleoside kinases and 5'-nucleotidases, and the relative rates of nucleoside triphosphate extracellular release. A cross talk between extra- and intra-cellular nucleoside metabolism exists, in which released nucleoside triphosphates, utilised as neuroactive signals, are catabolised by a membrane bound ectonucleotidase cascade system to their respective nucleosides, which are uptaken into brain cytosol, and converted back to nucleoside triphosphates by the salvage enzymes. Finally, phosphorolysis of brain nucleosides generates pentose phosphates, which are utilised for nucleoside interconversion, 5-phosphoribosyl-1-pyrophosphate synthesis, and energy repletion. This review focuses on these aspects of brain nucleoside metabolism, with the aim of giving a comprehensive picture of the metabolic network of nucleosides in normoxic conditions, with some hints on the derangements in anoxic/ischemic conditions.