Is Ecto-nucleoside Triphosphate Diphosphohydrolase (NTPDase)-based Therapy of Central Nervous System...

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Is Ecto-nucleoside Triphosphate Diphosphohydrolase (NTPDase)-based Therapy of Central Nervous System Disorders Possible? Katarzyna Roszek* and Joanna Czarnecka

Department of Biochemistry, Faculty of Biology and Environment Protection, Nicolaus Copernicus University in Torun, Poland

Abstract: Extracellular nucleotides and nucleosides are signalling molecules acting in all tissues and organs, including the central nervous system (CNS). A wide variety of effects, exerted by ecto-purines, requires that their levels, and ATP in particular, must be precisely controlled. Under physiological conditions, concentration of ecto-purines is regulated by a complex cascade of ecto-enzymes, including ecto-NTPDases (nucleoside triphosphate diphosphohydrolases), ecto-NPPs (nucleotide pyrophosphohydrolases/phosphodiesterases), ecto-alkaline phosphatases, and ecto-5’nucleotidase. Adenylate kinase, transferring the phosphate moiety between nucleotides, also plays a role in controlling ecto-purines concentration. Disturbances in the elements of purinergic pathway within the CNS underlie the induction and amplification of many neurological pathologies. ATP released in bulk from the cells, and not degraded by less efficient or dysfunctional ecto-nucleotidases, triggers excitotoxic damage and neuro-inflammation in the brain tissue. High ATP concentration activating specific receptors has been shown to be involved in various disorders throughout CNS, including brain injury and ischemia, neuro-inflammation, epilepsy as well as neuropathic pain and migraine. Taking the above mentioned influence of ATP into consideration, the modulation of ecto-NTPDase activity or its site-targeted delivery seems a good therapeutic method. The availability of effective brain-targeted drug-delivery system is one of the most significant challenges facing potential NTPDase-based treatment of CNS disorders. The application of genetically engineered stem cells as carrier vehicles offers a promising strategy for the efficient delivery of the enzyme to CNS tissues.

Keywords: ATP, ecto-NTPDase, ecto-nucleotidases, neurological disorders, purinergic signalling.

1. INTRODUCTION

The concept of purinergic signalling was proposed by Geoffrey Burnstock in the year 1972, and it initially referred to non-adrenergic and non-cholinergic (NANC) neurotransmission [1]. ATP was the molecule that fulfilled the criteria of a NANC neurotransmitter and it is now well recognized as a co-transmitter in all nerve types in both the peripheral and central nervous systems. Presently, it is a well recognized and widely accepted fact that purinergic signalling is involved in regulating the functions of all tissues and organs. In addition to ATP, guanine and uracil nucleotides/nucleosides may also act as signalling molecules, as described in literature [2-4]. However, in this article, we focused on ATP, its metabolic conversion and its role in the central nervous system tissues.

Extracellular purines activate two classes of the receptors: P1-adenosine receptors and P2-nucleotide (ATP and ADP) receptors in a concentration-dependent manner. P2 receptors are subdivided into P2X and P2Y families [5, 6]. The concentration of purines outside the cell depends on the balance between their release from the cells, re-uptake, and extracellular metabolism [5]. Ecto-nucleotides and

*Address correspondence to this author at the Department of Biochemistry, Faculty of Biology and Environment Protection, Nicolaus Copernicus University, Gagarin St. 7, 87-100 Torun, Poland; Tel: +48 56 6114501; Fax: +48 56 6114772; E-mail: [email protected]

ecto-nucleosides concentrations are controlled by a complex cascade of ecto-enzymes, including ecto-NTPDases (nucleoside triphosphate diphosphohydrolases), ecto-NPPs (nucleotide pyrophosphohydrolases/ phosphodiesterases), ecto-alkaline phosphatases, and ecto-5’nucleotidase [7-10]. The nucleotide kinase group of ecto-enzymes is also involved in the regulation of ecto-nucleotide concentration by transferring the phosphate moiety between nucleotides. ATP and ADP hydrolysis to AMP followed by its subsequent degradation to adenosine leads to the termination of a nucleotide signal [7, 8, 11].

In the central nervous system (CNS), the level of ecto-purines, and ATP in particular, must be precisely controlled. Extracellular ATP is released by exocytosis as well as by other mechanisms involving ATP binding cassette transporters, hemichannels, P2X7 receptors, or volume-sensitive chloride channels [12]. The high extracellular ATP concentration activating specific receptors is involved in a variety of CNS morbidities covering brain injury and ischemia, neurodegenerative diseases involving neuro-immune and neuro-inflammatory reactions, as well as neuropathic pain and migraine [1]. There is a continuously growing interest in the therapeutic potential of the compounds acting as purinergic signaling regulators (including receptor agonists and antagonists, ecto-enzyme inhibitors and activators, and nucleotide/nucleoside transport regulators) in a wide range of pathological states [1, 6, 13]. There is some experimental data available regarding the role of NTPDase1 in controlling

Katarzyna Roszek

6 Mini-Reviews in Medicinal Chemistry, 2015, Vol. 15, No. 1 Roszek and Czarnecka

ATP levels in cultured microglia, or in a cellular model of sympathetic neurons [14, 15].

However, it must be remembered that the ecto-nucleotidases activity may be altered under pathological conditions. It prompted us to explore the issue of nucleotides metabolizing ecto-enzymes as targets for potential activators or as therapeutic molecules themselves. In this article, the emphasis is placed on the contribution of ecto-NTPDases, particularly ecto-NTPDase1, in order to therapeutically decrease pathological ATP levels in CNS disorders.

2. ELEMENTS OF ADENINE NUCLEOTIDE SIGNAL-LING PATHWAY IN THE CENTRAL NERVOUS SYSTEM

2.1. Adenine Nucleotides and Nucleosides

Extracellular adenine nucleotides and nucleosides are signalling molecules involved in a wide range of actions in all the known tissues and organs, including the nervous system. In addition to short-term purinergic signalling in neurotransmission, neuromodulation, secretion, and platelet aggregation, it is now recognised that long-term (trophic) purinergic signalling is involved in cell proliferation, differentiation and death during development and regeneration, for example during neurite extension and neural stem cell differentiation [16].

Along with the massive nucleotide leakage upon cell damage, nucleotides can appear in the external milieu via various non-lytic pathways. The pathways include electro-diffusion through ATP release channels, facilitated diffusion by nucleotide-specific transporters or release in a Ca2+-dependent manner via vesicular exocytosis. Various excitatory and secretory tissues such as nerve terminals and chromaffin cells, pancreatic acinar cells and circulating platelets store ATP and ADP, together with other neurotransmitters and extracellular mediators. The storage occurs in specialized granules called synaptic vesicles, chromaffin granules or dense core granules [12].

In the nervous system, ATP is released from both peripheral and central neurons as well as from glial cells in response to mechanical deformation, hypoxia or agents such as acetylcholine and thrombin [1, 10, 17, 18]. While ATP contributes to the fast synaptic transmission via activation of ionotropic P2X receptors as well as neuromodulation via metabotropic P2Y receptors, ADP stimulates only P2Y, and adenosine stimulates only P1 receptors, thereby adjusting neuronal performance – (Fig. 1). Glial cells are often recipients as well as the source of extracellular ATP. Hence, purinergic neuron-glia signalling contributes bi-directionally towards information processing in the nervous system, involving sensory organs and brain areas, and computing sensory information [19].

The extracellular concentration of adenine nucleotides in different tissues is usually maintained at the level of approximately 10-7-10-9 M [20], whereas intracellular concentrations of nucleoside tri- and diphosphates stored in specialized granules are in milimolar range [21]. Physiological AMP and adenosine concentrations reach values of 10−6 - 10−5 M [20]. The most profound effect of

adenosine in the nervous system is inhibitory modulation of cellular activity and neurotransmitter release, and this effect is elicited at normal physiological levels. The extracellular concentration of adenosine in the brain was precisely determined to be 30-50 nM, based on an in vivo cortical cup technique. In vivo microdialysis techniques later led to an estimate of 180-270 nM in the basal forebrain and thalamus, 40-210 nM in the striatum and 109 nM in the cortex [22]. The highest adenosine concentrations, as expressed in picomoles in mg wet weight, are present in the cochlear nuclei, vestibular nuclei, and cerebellar cortex [21]. Ambient levels of brain adenosine appear to be largely under the control of astrocytes. Adenosine can appear in the extracellular milieu either via direct release of endogenous nucleoside or as a result of ATP/ADP breakdown via sequential ecto-nucleotidase reactions. Even a small decrease in NTPs and NDPs levels and/or changes in the enzymatic activity of the nucleotide/nucleoside metabolizing enzymes may cause a dramatic alteration in the nucleoside levels.

2.2. Purinergic Receptors

Purinergic receptors were first defined in 1976 and 2 years later, based on experimental data, two types of receptors were identified and proposed as P1 (for adenosine) and P2 (for ATP/ADP or other tri- and diphosphonucleotides) [16]. Four subtypes of P1 receptors were cloned, namely A1, A2A, A2B and A3. All the P1 adenosine receptors are typical metabotropic (G protein-coupled) receptors with specific agonists and antagonists available for each subtype. The expression level of P1 adenosine receptors in the human brain is region-specific. Moreover, age-related changes in adenosine receptor distribution were described in the brains of both animals and humans. It was observed that A1 receptor density decreased and A2A receptor activity increased with age [21].

In 1985, P2 receptors were subdivided into P2X and P2Y subtypes on the basis of their structure, course of action, and pharmacology [23]. Currently, we recognise the P2X receptor family as ligand-gated ion channel receptors and the P2Y receptor family as G protein-coupled receptors. Seven subtypes of the P2X and eight subtypes of the P2Y receptors were cloned and functionally characterised (see Table 1). The stoichiometry of these receptors is shown to involve three subunits, which form a functional trimer. In the central nervous system, cells heteromultimers (for example P2X2/3 receptors in nodose ganglia, P2X4/6 receptors in CNS neurones or P2X2/6 receptors in the brain stem) as well as homomultimers are involved in forming the trimer ion pore. Only P2X7 receptors do not form heteromultimers and P2X6 receptors will not form a functional homomultimer [5, 24].

Metabotropic P2Y receptors couple to form single heterotrimeric G proteins, although P2Y11 can couple to both Gq/G11 and Gs. P2Y12 however couples to Gi. P2Y receptors may form homo- and heteromultimeric assemblies, for example, P2Y1 receptors were shown to form a heteromeric complex with adenosine A1 receptors. Some P2Y receptors are activated principally by nucleotide diphosphates (P2Y1, P2Y6, P2Y12, P2Y13), while others are activated mainly by nucleotide triphosphates (P2Y2, P2Y4). Some P2Y receptors are activated by both purine and pyrimidine nucleotides

NTPDase-based Therapy of CNS Disorders Mini-Reviews in Medicinal Chemistry, 2015, Vol. 15, No. 1 7

(P2Y2, P2Y4, P2Y6), and others by purine nucleotides only (P2Y1, P2Y11, P2Y12, P2Y13). In response to nucleotide activation, recombinant P2Y receptors either activate phospholipase C and release intracellular calcium or affect adenylate cyclase and alter cAMP levels [25].

Majority of the purinergic receptor subtypes are widely expressed and distributed throughout the central and peripheral nervous system (Table 1). Under physiological conditions, they are generally considered to be involved in bi-directional neuronal–glial communication, exerting long-term effects on proliferation, differentiation, and migration. CNS astrocytes express P2X1-5 and P2X7 receptors. They also express P2Y1, P2Y2, P2Y4, P2Y6, P2Y12 and P2Y14 receptors as well as functional adenosine receptors. Microglial cells are characterized by the predominant expression of P2X4 and P2X7 receptors, but they also express P2Y1, P2Y2, P2Y4, P2Y6 and P2Y12 receptors. Oligodendrocytes progenitors are characterized by the functional expression of P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y13 receptors [26].

In neurons, P2X3 receptors were identified in rat midbrain synaptic terminals as well as in the central terminals of dorsal root ganglion neurons in the dorsal horn of the spinal cord. Other P2X receptors, such as P2X2 and P2X4, are widely distributed in neuronal structures, including the

cortex, hippocampus, cerebellum, and spinal cord. Moreover, P2X7 receptors have been identified in presynaptic terminals and axonal growth cones. There is strong evidence that P2X7 receptors influence neurons activity and they may coordinate microglial and neuronal/astroglial responses, both under physiological and pathological conditions [27-29]. Among P2Y receptors, P2Y1 are dominantly expressed in neurons, while P2Y2 receptors are also expressed at lower levels in all regions. In addition, P2Y4, P2Y6, and P2Y11-14 receptors have been found to be expressed in several types of neurons [26].

2.3. Ecto-enzymes Metabolizing Adenine Nucleotides/ Nucleosides

Extracellular adenine nucleotides and nucleosides concentrations are precisely controlled by the complex enzymatic cascade constituted by cell surface-located enzymes named ecto-nucleotidases, including ecto-NTPDases (nucleoside triphosphate diphosphohydrolases), ecto-NPPs (nucleotide pyrophosphohydrolases/ phosphodiesterases), ecto-alkaline phosphatases, and ecto-5’nucleotidase [7-10, 20]. Adenylate kinase from the transferases class also participates in the conversion of adenine nucleotides (Fig. 1).

The protein family of NTPDases hydrolyses nucleoside 5’-triphosphates and nucleoside 5’-diphosphates within

Table 1. Purinergic receptors and their distribution in the nervous system [compiled from: 1, 21, 25, 26].

Receptor Signal transduction mechanism Distribution in the nervous system

A1 Gi/G0, ↓cAMP Neurons of parietal, temporal, and occipital cortex, astrocytes

A2A Gs, ↑cAMP Brain neurons, astrocytes

A2B Gs, ↑cAMP Uniformly distributed

P1

A3 Gi/G0, Gq/G11, ↓cAMP, PLC-β activation Neurons of cerebellum and hippocampus, astrocytes

P2X1 Intrinsic cation channel (Ca2+ and Na+) Cerebellum, dorsal horn spinal neurons, astrocytes

P2X2 Intrinsic ion channel (Ca2+) CNS neurons, autonomic and sensory ganglia, astrocytes

P2X3 Intrinsic cation channel Sensory neurons, nucleus tractus solarius neurons, sympathetic neurons, astrocytes

P2X4 Intrinsic ion channel (Ca2+) CNS neurons, astrocytes, microglia

P2X5 Intrinsic ion channel Spinal cord, astrocytes

P2X6 Intrinsic ion channel CNS neurons, motor neurons in spinal cord

P2X

P2X7 Intrinsic cation channel and a large pore with prolonged activation Neurons, astrocytes, microglia, apoptotic cells

P2Y1 Gq/G11, PLC-β activation Neurons and glial cells

P2Y2 Gq/G11 and possibly Gi/G0, PLC-β activation Astrocytes

P2Y4 Gq/G11, PLC-β activation Brain neurons and glial cells

P2Y6 Gq/G11, PLC-β activation Activated microglia

P2Y11 Gq/G11 and Gs, PLC-β activation Brain neurons and oligodendrocytes

P2Y12 Inhibition of adenylate cyclase Glial cells, spinal cord

P2Y13 Gi/G0 Brain neurons and oligodendrocytes

P2Y

P2Y14 Gi/G0 Discrete brain regions


different tissues and cells. Eight different members of the NTPDase family (NTPDases 1-8) have been discovered, cloned, and functionally characterized. Despite their wide substrate specificity, all NTPDases are able to hydrolyse adenine nucleotides [8, 9, 20]. Four of them – NTPDase 1, 2, 3, and 8 - are expressed as cell surface-located enzymes that possess similar amino acid sequences and membrane topology with two membrane spanning domains and catalytic sites facing the extracellular matrix [30]. NTPDases 5 and 6 exhibit intracellular localization and undergo secretion after heterologous expression while NTPDases 4 and 7 are entirely intracellularly located, facing the lumen of cytoplasmic organelles.

All the cell surface members of ecto-NTPDase family are highly glycosylated proteins with molecular masses about 70-80 kDa and close immunological cross-reactivity. The enzymatic proteins contain two predicted transmembrane domains connected with a large extracellular loop containing central hydrophobic region with five highly conserved sequence domains known as “apyrase conserved regions” (ACR) [8]. Ecto-NTPDases have an alkaline optimum pH. Milimolar concentrations of either Ca2+ or Mg2+ can stimulate the catalytic activity of enzymes classified as NTPDases. These enzymes may exist either in monomeric or in homo-oligomeric (dimeric to tetrameric) states [11]. NTPDase1 (also known as CD39) hydrolyses ATP and ADP equally well while NTPDases 2, 3, and 8 prefer to hydrolyse ATP over ADP [8, 20].

Histochemical investigations demonstrated the hydrolysis of nucleoside tri- and diphosphates in all the cell types of the nervous system [30]. Ecto-NTPDases 1, 2, and 3 are expressed in the mammalian brain where they mediate the termination of ATP signalling while ecto-NTPDase 8 expression is very low in the brain [31]. Ecto-NTPDase1 is localized at the surface of endothelial cells in the CNS vessels and is strongly expressed in microglia while it is not expressed by neurons and astrocytes [32]. Ecto-NTPDase 2 is associated with progenitor cells in the adult rodent brain

[33] and is expressed in muscularised vessels [34], cultured astrocytes [35], non-myelating Schwann cells, and other glial cells of central and peripheral nervous systems [8]. Ecto-NTPDase 3 is expressed in multiple brain regions and at the surface of PC12 cells as the predominant ecto-nucleotidase [36]. Immunohistochemical studies revealed that this enzyme is strongly associated with axon-like neuronal structures in the brain, where it may act as a main regulator of extracellular ATP levels.

Ecto-NPPs play multiple physiological roles, including nucleotide recycling, the modulation of purinergic receptor signalling, and regulation of extracellular pyrophosphate levels. Although the seven distinct NPP-encoding genes were described, only three enzymes (NPP1-3) are capable of hydrolysing various nucleotides and are therefore relevant in the context of the purinergic signalling cascade. These members show significant (40-50%) sequence similarities at the protein level and are classified as type II transmembrane glycoproteins. The proteins consist of an intracellular N-terminal domain, a single transmembrane domain and a large extracellular domain with a catalytic site, and a putative C-terminal “EF-hand” motif [37]. NPP6–7 hydrolyse only phosphodiester bonds in lysophospholipids or other choline phosphodiesters while natural substrates for NPP4–5 still remain unknown [20]. NPP activity shows strong alkaline pH optimum, dependence on divalent cations, and it is inhibited by glycosaminoglycans. Notably, an unambiguous distinction between the members of NPP and NTPDase families is often complicated by their co-expression to a variable extent among the mammalian tissues and shared similarities in substrate specificity.

Human NPP1 is highly expressed in bone and cartilage tissues, whereas an intermediate enzyme expression is found in the heart, liver, placenta, kidney, and testis. In CNS, NPP1 is expressed in the capillaries of the brain and in rat C6 glioma cells, whereas it is not detected in neurons or glia [10]. NPP2 is a secreted lysophospholipase D that hydrolyses albumin bound or membrane-derived lysophosphatidylcholine

Fig. (1). Metabolism of extracellular adenine nucleotides and nucleosides and their affinity to the purinergic receptors.


(LPC) to produce equimolar amounts of lysophosphatidic acid (LPA) and choline [38]. A splice variant of NPP2a was identified during intermediate stages of the rat brain oligodendrocyte differentiation and myelin formation. Thus, the production of LPA by NPP2 might be important for cerebral maturation. In addition, the mRNA NPP2 expression was detected in the hippocampus, cerebral cortex, olfactory bulb, cerebellum, and striatum throughout the development [39]. NPP3 is expressed in choroid-plexus epithelial cells and may contribute to the secretion of the cerebral spinal fluid. NPP1 mRNA increases through maturation, whereas NPP3 was demonstrated to decrease with age [39].

Alkaline phosphatases (APs) are non-specific phosphomonoesterases that release inorganic phosphate from several organic compounds and degrade nucleoside 5’-triphosphates, diphosphates, and monophosphates. The mammalian APs are membrane-bound and present an optimal activity at the alkaline pH values. Three isozymes are tissue-specific and 90-98% homologous, i.e. the intestinal AP, placental AP, and germ cell AP, while the fourth isozyme, the tissue nonspecific alkaline phosphatase (TNAP), is approximately 50% identical to the other three isozymes and abundantly expressed in the bone, liver, kidney and, at lower levels, in some other tissues [20]. TNAP is anchored to the plasma membrane through a glycosyl-phosphatidylinositol (GPI). Except for the recognized and essential role in osteogenesis, the role of TNAP in other organs, including the central nervous system, remains unknown [10].

Studies demonstrated that the most prominent catalytic activity of TNAP in the nervous system is associated with the endothelium of brain capillaries, the choroid plexus, and the meninges [40]. It was also demonstrated that TNAP is found in the neuropil of many brain regions, including the olfactory bulb, septum, thalamus and hypothalamus, cerebral cortex, tegmentum, and dorsal and ventral medulla [40]. The AP activity detected in the brain tissues results from the expression of TNAP both in endothelial and neuronal cells.

Ecto-5’-nucleotidase - seven human 5'-nucleotidases were isolated and characterised to date, with five of the enzymes located in the cytosol, one in the mitochondrial matrix, and one attached to the plasma membrane [20]. Surface-associated ecto-5'-nucleotidase (ecto-5’NT, also known as CD73) efficiently hydrolyses 5'-AMP and shows no activity towards nucleoside 2'- and 3'-monophosphates. The formation of adenosine from extracellular AMP and the subsequent activation of P1 adenosine receptors is one of the most important actions of ecto-5’-nucleotidase [10]. The mammalian ecto-5’NT consists of two glycoprotein subunits with apparent molecular masses of 60–70 kDa and is anchored to the plasma membrane at the C-terminus by glycosyl-phosphatidylinositol (GPI) [21].

Ecto-5'-nucleotidase is expressed in different tissues, with high expression level in the colon, kidney, brain, liver, heart, and lung [20]. Ecto-5’-nucleotidase is abundant in synaptic membranes from the hippocampus, cerebellum, and medulla oblongata [41]. Most human brain areas demonstrate intermediate activity of ecto-5’NT (for example the parietal lobe, cingulate cortex, insula, caudate nucleus,

putamen, thalamus (anterior), subthalamic nucleus, nucleus ruber, substantia nigra, amygdala, and hypothalamus [21]. Age-related alterations were also observed for ecto-5’-nucleotidase activity from CNS. The enzyme activity was 5-fold higher in the hippocampuses of aged rats compared to young rats [42].

Adenylate kinase belongs to the nucleoside monophosphate kinases family (NMPK). Originally, AK was defined as ATP-AMP phosphotransferase that catalyzes the reversible reaction of a high-energy β- and γ-phosphate groups transfer between adenine nucleotides. However, most of adenylate kinases, classified on the basis of their sequence and structural similarity, use also other ribonucleotides and deoxyribonucleotides as acceptors and donors of the phosphate residue. There are nine isoforms of human adenylate kinase (AK1-AK9) distinguished in the literature [43, 44]. Adenylate kinases are composed of a single polypeptide chain, and only a few cases of homo-oligomerization were reported [45, 46]. Most isoforms of the enzyme show intracellular localization, but only the soluble exo-AK or cell surface-located ecto-AK may be one of the elements of the nucleotide signalling system [47, 48]. The presence of ecto-adenylate kinase on the surface of neurons in the brain synaptosomal fractions and model PC12 cells suggests the involvement of this enzyme in the regulation of the CNS function [49-51].

3. THE CENTRAL NERVOUS SYSTEM DISORDERS

Due to multiple physiological functions of the purine molecules signalling in the central nervous system, disturbances in the elements of purinergic pathway underlie the induction and amplification of many pathologies. The high concentration of ATP released from the cells and not degraded by less efficient or dysfunctional ecto-nucleotidases triggers excitotoxic damage and neuroinflammation in the brain tissue (Fig. 2). Following various CNS insults, the high level of ecto-ATP is sufficient to activate P2X7 receptors and leads to oligodendrocytes demyelination, axonal damage, and cell death [29]. Eventually, it activates the immune response of glial cells: astrocytes, microglia and Schwann cells [26, 52]. In this chapter, we describe some CNS disorders and the involvement of adenine nucleotides/ nucleosides signalling in their initiation and/or development.

3.1. Ischemia and Hypoxia - Lack of Oxygen Induces ATP Release

Metabolism of the neurons requires an intensive local blood flow to supply them with oxygen and energetic materials. On the other hand, blood is the main recipient of metabolites from the brain to the tissues located outside. Ischemia and hypoxia in the brain are sudden and acute pathological conditions connected with the oxygen deprivation.

The effects of ischemia and hypoxia are particularly acute because the brain has an extremely high respiratory quotient (close to 1), which means that the whole pool of brain oxygen, and up to 25% of the oxygen uptake in the lungs, is consumed by glucose metabolism. Moreover, brain has a very high energy demand and almost complete lack of


carbohydrates reserves. Under normal oxygenation of the brain, the energy demand is met by the breakdown of ATP produced in glycolysis. Moderate hypoxia causes a generalized reduction in neuronal excitability and the decrease in intracellular ATP concentration throughout the central nervous system. With longer ongoing hypoxia, the amount of energy substrates is lowered, ATP is degraded, and the ion pumps are stopped. Lactate is formed as a result of the anaerobic metabolism of glucose and followed by intracellular acidosis. After stopping the sodium-potassium pump (Na+/K+-ATPase) and calcium pump (Ca2+-ATPase) the ion homeostasis is disturbed and initiates persisting depolarization. High intracellular concentrations of calcium ions trigger the formation of free radicals and peroxides, the activation of proteases, nucleases, and phospholipases, i.e. processes leading to the neuronal degradation. Free radicals and cyclooxygenase COX-2 activation gives rise to pro-inflammatory factors that activate microglia and leukocyte adhesion. Moreover, the cells start swelling and plasma components flow to the extracellular space. All these processes leading to the death of neurons are denoted as excitotoxicity [53-55].

The conditions of severe hypoxia triggered by the reduction of RCBF (regional cerebral blood flow) below 0.2ml/100g/min lead to both excitatory (glutamate and aspartate) and inhibitory (GABA, glycine) neurotransmitters release. At the same time the oxygen deprived astrocytes exhibit lower efficiency of glutamate uptake from synaptic space. Uncontrolled neuronal depolarization and the

prolonged time of their activation is the consequence of excessive stimulation of glutamate receptors [54].

In addition to glutamate-mediated hyper activation, enhanced ATP signalling is also observed during ischemia. It was shown that, after oxygen and glucose deprivation, oligodendrocytes release ATP through the opening of pannexin hemichannels [56]. Ischemic conditions change the P2 receptor profile: the expression of P2X2 and P2X4 receptors is up-regulated in in vitro cultures of the hippocampus, cortex and striatum cells. P2X7 receptors are up-regulated on neurons and glial cells in the rat cerebral cortex [1, 57].

The consequences of P2X receptors activation can be reduced by ATP degrading NTPDase1, or by receptor antagonists, and blockers of pannexin hemichannels which release ATP [1, 56]. ATP hydrolysis by NTPDase1 and 5’-nucleotidase leads to the subsequent production of adenosine which is known for its immunosuppressive and neuro-protective activity. Adenosine levels rise markedly in response to ischemia, hypoxia, excitotoxicity, or inflammation playing a role of neuro-protectant under these conditions. However, adenosine may also contribute to the neuronal damage and cell death under other circumstances. There are some recently published papers covering this topic in more detail [58-60].

3.2. Neuroinflammation – Lower is Better

Inflammatory reactions follow any brain damage. The CNS acute injuries (such as stroke and trauma), infections,

Fig. (2). High ATP concentration induces various pathological conditions in the central nervous system.


neurodegenerative and oncogenic disorders initiate a prolonged inflammatory response involving microglial activation and infiltration of macrophages and neutrophils. Interaction between various components of the brain immune system during their activation occurs by multiple signalling molecules, including nucleotides [61].

Under stressful conditions, ATP is released by several cell types to the extracellular space in an uncontrolled (necrosis) or controlled (pannexin hemichannels) manner [62, 63]. The elevated concentration of extracellular ATP in the local microenvironment of the injury marks the damaged site and contributes to the promotion of a primary immune response. The modulation of an inflammatory response by extracellular ATP results from its specific influence on both immune and non-immune cells through the activation of P2 receptors [61].

The P2X7 receptor activated by milimolar ATP concentration is the most frequently mentioned P2 receptor involved in the brain inflammatory processes. This type of receptor is widely distributed on the surface of brain cells. Besides microglia and astrocytes, it has also been found on some populations of neurons [64] and oligodendrocytes [3, 65]. In the first stage of neuroinflammation, extracellular ATP, via P2X7 receptors, functions as a proinflammatory and immunostimulatory mediator in the microenvironment of damaged cells. The activation of P2X7 receptors on microglia cells increases the production of pro-inflammatory interleukins: IL-1α, IL-1β, IL-6, IL-18 as well as TNFα and leukemia inhibitory factor (LIF), triggering the inflammatory response [61]. It was shown that blocking P2X7 receptor in the hippocampal slice cultures strongly decreases IL-1β levels produced after LPS treatment [65]. Microglia activation subsequently leads to the release of ATP from astrocytes. The activation of both P2X7 and P2Y1 or P2Y2 receptor subtypes on astrocytes initiates astrogliosis, enhanced cell proliferation, excessive growth, and hyperplasticity. These processes isolate the damaged brain area from the surrounding healthy cells and lead to synthesizing neurotrophins and pleiotrophins that participate in the neuronal recovery [55, 66, 67].

In the second stage of inflammation, the role of ATP appears to be rather immunomodulatory and anti-inflammatory. Ecto-enzymes, mediating the hydrolytic cascade, and decrease in the nucleotides concentrations, plays a key role at this stage. Varying levels of nucleotides regulate the profile of activated P2 receptors. In rat microglia cultures, low micromolar ATP concentrations inhibit the production of pro-inflammatory cytokines, presumably through the P2Y receptor activation and increase in the intracellular Ca2+ level [68]. Moreover, nanomolar ATP concentrations released after the LPS-stimulation of the rat microglia induced anti-inflammatory IL-10 expression by the autocrine activation of P2Y receptors [69]. Similar processes were observed outside the central nervous system. Micromolar concentrations of ATP markedly reduced the inflammatory reaction by simultaneous inhibition of TNF-α and stimulation of IL-10 release in the LPS-stimulated human blood [70, 71]. The contrasting effects of ATP-mediated P2 receptor activation on TNF-α release were reported by Kucher and Neary. In the LPS-stimulated

astrocytes P2X receptor activation attenuates, whereas P2Y receptor activation potentiates TNF-α release. These observations suggest that astrocytes can sense the severity of damage in the CNS via ATP released from damaged cells, and therefore, modulate TNF-α mediated inflammatory response, depending on extracellular ATP concentration and corresponding type of astrocyte P2 receptor activated [72].

Over-activation of the immune system may lead to uncontrolled or chronic inflammation, resulting in the collateral cell damage and destruction of the healthy tissues. While the short-term ATP-mediated activation of the P2X7 receptor can lead to the proliferation of microglia, the long-term stimulation of the immune cells can direct them to apoptosis [52, 66]. Therefore, inflammatory and immune responses must be tightly regulated [61, 73]. The progressive adjusting of immune response repertoire is achieved by changes in the expression profile of purinergic receptors as well as ecto-enzymes on the immune cells involved in inflammatory reactions. Two ecto-enzymes: NTPDase1 (CD39) and 5’-nucleotidase (CD73) efficiently control extracellular nucleotide concentrations and thereby regulate the extent of purinergic signals, as evidenced by several studies using ecto-enzyme knock-out models [74-79]. However, it must be remembered that the ecto-nucleotidase activity may be altered under pathophysiological conditions.

3.3. Pathologies of Adenine Nucleotide Signalization System in Chronic Neurodegenerative Disorders

Neurodegenerative diseases are a group of chronic, progressive disorders characterized by the gradual loss of neurons in diverse areas of the central nervous system. Neuroinflammation has been often implied in the diseases progression [80]. Although the role of nucleotides in chronic neurodegenerative disorders is not quite clear, it appears that they aggravate the consequences of the primary disease [67].

Parkinson disease (PD) is a neurodegenerative disease that is characterized by the degeneration of dopaminergic neurons (expressed in accumulation of Levi's Bodies and cell death) of the substantia nigra. It is believed that chronic inflammation and neuroinflammation in combination with genetic background may act as a crucial environmental stress factor to promote progressive degeneration of neurons [81]. Microglial and astroglial cells, activated by any compatible initial factors, contribute to the propagation of inflammatory processes and promote pathologies characteristic for PD: the selective degeneration of nigral dopaminergic (DA) neurons, formation α-synuclein aggregates in the spared DA neurons, and consequently locomotor deficits [81, 82].

Purinergic signalling is strongly associated with striatal neuronal cell function and PD pathogenesis. Different types of P2 receptors are expressed in dopaminergic system cells and co-localization of the dopamine-(D1) receptor with the P2X1 receptor in the rat brain is also known [83]. In PC12 pheochromocytoma cells (a frequently used model of sympathetic neurons), ATP and related nucleotides induce a release of dopamine, but on the other hand, they decrease the electrically evoked release of endogenous dopamine in rat neostriatum [84, 85]. The loss of dopaminergic cells in the substantia nigra correlates with presence of activated


microglia cells and indicates the involvement of ecto-ATP in the etiology of self-inducing, destructive, and uncontrolled local inflammation [86]. It was observed that P2X7 receptors are highly expressed in the cells in dopaminergic areas affected by PD [87-89]. The release of milimolar ATP concentrations from damaged neuronal cells can also trigger cell death in the surrounding cells that contain P2X7 receptor on their cell surface. This leads to the amplified cell necrosis [1]. Additionally, ATP may control the striatal cell damage and modulate neuronal firing through the activation of P2X2, P2X4 and P2X6 receptors [90].

Currently, researchers propose a few therapeutic strategies for PD treatment. Most commonly prescribed is the symptomatic therapy with the use of dopamine precursor L-DOPA, which causes long-term treatment problems such as dyskinesia and autonomic or psychotic dysfunction [91]. Despite high expectations, trials with standard non-steroidal anti-inflammatory drugs do not give the expected results [92, 93]. The new therapeutic directions may involve immunological therapies, assuming the dysfunction and abnormal infiltration of peripheral immune cells through the blood-brain barrier of PD patients [94, 95]. The infiltration of peripheral T-cells from the systemic circulation leads to their recruitment to the substantia nigra and provides neuroprotective agents to pathologically changed neurons (the so called “Trojan Horse Therapy”) [95]. Still, there is no treatment available to stop the early neuroinflammatory and neurodegenerative processes, therefore the regulation of nucleotide signalling associated with ecto-nucleotidase-mediated lowering of the ATP concentration seems to be a step in the right direction.

Alzheimer's disease is defined by specific neuropathology: the aggregation of β-amyloid proteins (AβPs) in the form of an extracellular neuritic plaque and neocortical neurofibrillary tangles (NFTs). Implications of AβPs and NFTs presence are not fully clear since they were also found in healthy brains. On the other hand, the severity of cognitive impairment in AD correlates best with the neocortical neurofibrillary tangles [96, 97]. Moreover, the AβPs and other plaque substances are neurotoxic. Together with the damaged neurons and neurites they participate in the discrete and micro-localized stimulation of inflammation. The cumulated, chronic damage by inflammatory mechanisms is likely to contribute to the progression of AD-connected neuronal degeneration [93].

Some researchers link Alzheimer's disease progression with purinergic signalling. It was shown that AβPs oligomers induced an increase in ATP release from astroglial cells [98]. The elevated ATP concentration participates in the initiation of the neuroinflammation through P2X7 receptors activation. The microglia from AD brains represent the 70% increase in P2X7 receptor protein expression as compared with non-demented brains. The receptor proteins co-localize in vivo with microglia after AβPs injection into the rat hippocampus [99]. The presence of AβPs also promoted the P2X4 receptor concentration on the surface of cultured neurons. It also induced a caspase-3-mediated cleavage of the receptor that slowed channel closure times and prevented the agonist-

induced internalization of the receptor [100]. P2Y1 receptors are expressed on a number of structures such as neurofibrillary tangles, neuritic plaques, and neuropil threads characteristic for Alzheimer’s disease [1]. On the other hand, the nucleotide signalling can participate in the prevention of AD-associated damage. It was shown, that the activation of the P2Y2 receptor by UTP stimulates α-secretase-dependent processing of the amyloid precursor protein (APP). Under these conditions, APP cleavage generates soluble, non-amyloidogenic sAPPα, which presents neurotrophic and neuroprotective activities. The data suggests that the UTP-induced release of sAPPα in the external milieu may promote neuronal viability [101].

The profile of the ATP signalling system elements is also altered in other neurological diseases e.g. amyotrophic lateral sclerosis (ALS) and autoimmunological multiple sclerosis (MS). Amyotrophic lateral sclerosis is characterized by the progressive degeneration of motor neurons in the spinal cord, brainstem, and motor cortex. ALS results from mutations in superoxide dismutase (SOD1) gene. This mutation causes astrocytes to display a neuroinflammatory and neurotoxic phenotype [1]. In the transgenic SOD1G93A mouse model, a significant increase in the expression of P2X1 receptor as well as enhanced P2X7 immunoreactivity was observed, indicating that the neuroinflammatory effects can be associated with ATP signalling. In SOD1G93A astrocyte cultures, the pharmacological inhibition of the P2X7 receptor or increased extracellular ATP degradation with NTPDase1 was sufficient to completely supress the damage towards motor neurons [102].

The demyelination process observed in multiple sclerosis seems to result from either autoimmunity or a dystrophy of the myelinating cell oligodendrocyte. Milimolar ATP concentration is sufficient to kill oligodendrocytes by activating P2X7 receptors present on their surface. The expression of the P2X7 receptor is also elevated in neurons and astrocytes of MS patients and in brain samples from experimentally demyelinated rodents [1]. Interferon�β (IFN�β) has beneficial effects in remitting/relapsing MS, perhaps by preventing astrocyte apoptosis. The levels of NTPDase1 and ecto-5′-nucleotidase increased in synaptosomes from the cerebral cortex of rats which were experimentally demyelinated and then treated with IFN�β, indicating that IFN-β might interfere with the metabolism of purines [103]. Patients with relapsing form of MS have a strikingly reduced number of NTPDase1-positive, immunosuppressive regulatory T cells, which suggests that ecto-nucleotidase activity may be involved in the remitting of MS [1].

Chronic neuroinflammation has been increasingly implicated in the initiation and progression of neurodegenerative diseases. It is hypothesized that any intervention that can interrupt the chronic inflammation process should be effective in either halting or slowing down the progression of the diseases. Treatments with multiple actions directed at inflammatory mechanisms, including the elevated ATP concentration and P2X7 receptor activation, might represent the most promising therapeutic strategies for neurodegenerative diseases [80].


3.4. Neuropathic Pain and Migraine

The participation in pain transmission was probably the first postulated function of ATP. Locally released ATP can initiate pain pathways through activation of P2X3 and P2X2/3 receptors located on sensory fibres in visceral organs. In the central nervous system, P2X3 receptors located on primary afferent nerve terminals in spinal cord play a significant role in neuropathic and inflammatory pain [1]. P2X2, P2X4 and P2X6 receptors located on the dorsal horn neurons spread nociceptive information further along the pain pathway. It is worth noticing, that pain is a normal signal which invokes the appropriate response to dangerous stimuli. The pathological state is associated with the excessive induction of pain, such as migraine or allodynia. According to Headache Classification Subcommittee of the International Headache Society, migraine is defined as recurrent seizures of headache accompanied by autonomic symptoms. In the past, the role of ATP in the etiology of migraine was associated only with the vascular theory and influence of ATP on blood vessels of muscles. Presently, more attention is paid to the neuronal dysfunction, mainly in the area of the trigeminal nerve and the thalamus. ATP released into the extracellular space contributes to headache by activating P2X3 receptors present on sensory neurons of the trigeminal ganglion [104]. The P2X3 receptor and other subtypes of P2 receptors involved in nociception (P2X2, P2Y1, P2Y2, P2Y4, P2Y6) are found in the neurons of “migraine area” [105, 106]. Moreover, it is observed, that ATP concentration in the cerebrospinal fluid rises significantly during migraine [107]. It is suggested that ATP in the cerebrospinal fluid may be involved in the activation and sensitization of P2X3 receptors on primary afferent nerves in trigeminal ganglia [1]. Besides the neuronal P2X3 receptor, two other subtypes of purinergic receptors, P2X4 and P2X7, localized on microglia cells are involved in the initiation of nociception. The P2X4 activation induces neuropathic pain by neuronal hyper-excitability due to ATP-initiated increase in Cl- concentration in spinal neurons [108].

3.5. Epileptic Seizures – Ongoing Therapeutic Problems

Epilepsy covers a diversified group of disorders caused by different cellular and molecular alterations of the brain, leading to recurrent synchronous and uncontrolled neuronal discharges (epileptic seizures). In human and animal models, status epilepticus results in the selective neuronal loss and gliosis, particularly within the hippocampus, as well as cognitive deficits and lasting hyper-excitability [109]. The increase in excitability results from the imbalance between the concentrations of inhibitory and excitatory neurotransmitters [110]. It is believed that the disturbances in the ecto-nucleotide metabolism may also lie in the background of epilepsy. Convulsive activity produces an overall reduction of ATP levels but intense activation of brain neurons triggers ATP release. Following status epilepticus, there is a prominent increase in the P2X7 receptor expression [111]. In epileptic patients, the hydrolysis of ATP to AMP is reduced, which can be a result of mutations in the NTPDase1 gene. Chromosomal position of human NTPDase1 gene coincides with the positions of the genes responsible for the focal epilepsy with auditory symptoms [111].

Reduced NTPDase activity leads to the increased levels of ATP outside the cell and decreases the concentration of neuroprotective ecto-adenosine [54]. The studies on the mice model confirmed the important role of adenosine and A1 receptors in protection against the acute and chronic epileptogenic effects [112]. The exact effect of ATP on neural activity depends on the actual activity of ecto-nucleotidases and the expression level of the purinergic receptors, both of which alter during epileptogenesis [113]. It is worth noticing that epileptic seizures, experimentally induced in healthy rats, caused a stable increase in the activity of NTPDase1, which degraded rising ecto-ATP levels and protected cells against excitotoxicity [114].

Up till now, anticonvulsant drugs and complementary therapies are ineffective in every third patient, so there is an urgent need for a novel treatment [1, 109]. As the effect of ATP on neural activity depends, among others, on the local NTPDase activity which may be altered during epileptogenesis, the increase in ecto-NTPDase activity seems a good therapeutic direction.

4. PURINERGIC THERAPIES

It must be remembered that purinergic regulation is not a single interaction but a space-time coincident event that explains the multidirectional nature of the information delivered and pleiotropic effects exerted by extracellular purinergic molecules. There is still growing evidence for a variety of pathological conditions, including neurological disorders, that result from altered purinergic mechanisms. Therefore, the aspect of “purinergic drugs” or drug targets, represents a promising field for the investigation of new therapies for the nervous system disorders. Current therapeutic strategies for CNS, as seen from clinical trials, focus rather on the use of P1 and P2 receptors antagonists [13]. Much effort is also made to identify potent and selective inhibitors for each member of the ecto-nucleotidases family [115, 116].

Since ATP is an excitatory neurotransmitter and its increased concentration underlies many diseases of the nervous system in the manner described in the previous section, we postulate that local up-regulation of ATP metabolism through ecto-NTPDases activation or delivery should be given more attention. Such experiments have recently been performed in vitro on healthy primary hepatocytes and HepG2 cells exposed to acetaminophen overdose. Acetaminophen elicited significant release of ATP to the extracellular environment at levels that were high enough to promote direct cytotoxicity. In the above experiments, the potato apyrase capable to hydrolyse nucleoside triphosphates and diphosphates (with high ATPase/ADPase ratio) was used. Apyrase treatment decreased ATP levels and Ca2+ mobilization from intracellular stocks, and therefore, reduced acetaminophen cytotoxicity [64]. The apyrase treatment was also effective in preventing neuronal loss and death induced by lipopolysaccharide or lipoteichoic acid in mixed neuronal/glial cultures [3]. The overexpression of ecto-NTPDase1 in PC12 cells (a cellular model of sympathetic neurons) resulted in the enhanced removal of exogenous ATP [15].


Removing ATP-dependent signalling in thermally injured tissues through NTPDase1-mediated ATP hydrolysis also reduced tissue necrosis in vivo. Necrosis measured with TNF-α and IFN-β expression was less extensive in the apyrase group when compared with the burn group at 24 hours and 5 days [117]. In mouse model of asthma, the treatment with apyrase markedly attenuated airway inflammation and reduced the number of inflammatory cells as well as the levels of cytokines in the bronchoalveolar lavage fluid [118]. The antithrombotic action of systemically applied apyrase or NTPDase1 also affects the ischemic brain and other disorders etiologically connected with circulatory system morbidities [119-121]. Application of recombinant NTPDases into the bloodstream of mice with induced stroke, effectively modulated thrombosis and reduced infarct size following transient middle cerebral artery occlusion. The side effects were less severe than in case of conventional anticoagulants [119, 120]. However, it should be noted that the blood-brain barrier is not permeable to intravascularly applied enzymes. We believe that the construction of apyrase/NTPDase delivery systems, targeted directly to the extracellular space of brain cells, will have particularly promising therapeutic potential.

Possible strategies to develop the NTPDase-based treatment of neurological disorders include searching for enzyme activating agents and conditions, recombinant NTPDase production and its site-targeted delivery, and stem cell-based therapies with cells genetically modified to express active NTPDase.

4.1. NTPDase Regulation

The modulation of ecto-nucleotidases activity under pathophysiological conditions may occur on both the gene expression and/or protein level. Although there are some literature reports on physiological and pathological stimuli capable of modifying the ecto-NTPDase activity [122], much more investigation is needed to highlight the importance of controlling these activities in pathological conditions in the nervous system.

Synaptosomes from the rat hippocampus and cerebral cortex are an example of important adaptive plasticity of the ecto-nucleotidase activity. Up-regulation of hydrolytic activity towards excessive ATP levels was observed after the induction of status epilepticus by pilocarpine or kainate [114]. Results obtained by Schmatz et al. [123] show that NTPDase and 5′-nucleotidase activities rise in synaptosomes from diabetic rats. The authors suggest that these activities are also related to an adaptive plasticity of the ecto-nucleotidase pathway that could occur in order to terminate the influence of extracellular ATP, including its cytotoxic effects, and to increase the production of extracellular adenosine, a known endogenous neuromodulator and neuroprotective agent. Additionally, treatment with resveratrol significantly increases NTPDase and 5 ′-nucleotidase activities in the cerebral cortex synaptosomes of healthy and diabetic rats [123].

The mechanisms of ecto-NTPDases transcriptional and post-translational regulation are not well known. Protein phosphorylation appears to be a probable mechanism of

NTPDase activity regulation. Both NTPDase1 and NTPDase2 contain potential phosphorylation sites with obvious implications for undergoing functional modification. Thus, NTPDase1 was detected as a phosphoprotein in the hippocampal slices, synaptic plasma membrane fragments, and cultured astrocytes from the rat brain [124]. The differential phosphorylation of NTPDase2 splicing variants leads to the modification of its activity. A study by Wang et al. shows that 100 µM 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeable analogue of diacylglycerol, and a protein kinase C activator, increases NTPDase2 (β isoform) activity by 26.4% [125].

Ecto-NTPDase1 activity may also be modulated by the inhibition of phosphodiesterase-3 (PDE3). RAW macrophages and human umbilical vein endothelial cells (HUVECs) were treated with the PDE3 inhibitors, namely cilostazol and milrinone, then analysed against NTPDase1 mRNA and protein levels. HUVECs expressed an elevated NTPDase1 protein level, while macrophage NTPDase1 mRNA and protein were both elevated after PDE3 inhibition. HUVEC NTPDase activity increases by 25% during the cilostazol and milrinone treatment. The ubiquitination of NTPDase1 decreases its activity by 43% when compared to controls. These results indicate that PDE3 inhibition regulates endothelial NTPDase1 at a post-translational level [126].

Other post-translational modifications relevant for NTPDase1 catalytic activity are glycosylation reactions that appear crucial for reaching the cell surface, and palmitoylation. These post-translational modifications appear to be constitutive and contribute to the integral membrane association of this ecto-nucleotidase in lipid rafts [for details see 8].

Possible activators of the ecto-nucleotidases are an evident way to increase the acivity of these enzymes. However, the presence of several ecto-nucleotidase family members with high structure similarity on one hand, and differences in the catalytic properties on the other hand, makes the development of specific and potent activators difficult and demanding. The structural data imply that, the protein structure rather than the amino acid sequence in conserved apyrase regions (ACRs), may be of major relevance in determining catalytic differences between these enzymes. Despite the successful crystallization of rat NTPDase1 and NTPDase2 as prototype members of ecto-nucleoside triphosphate diphosphohydrolase family, and a 3-dimensional modelling of potato NTPDase1 [9, 127, 128], the molecular mechanisms that account for the differences in catalytic properties between members of the ecto-nucleotidase family still remain unknown. However, the determination of an enzymatic protein structure in free and substrate-bound states by X-ray crystallography may be helpful in designing specific and potent modulatory molecules. Searching for nucleotidase activators is still at an early stage, but undeniably, it will be an area of research interest in the future.

4.2. NTPDase Delivery System

Different pathological conditions in the nervous system often decrease the local activity of ecto-nucleotidase at cell surface. NTPDase-based treatment of CNS disorders requires a


good source of well characterized, catalytically active enzyme. In most in vivo and in vitro experiments, the potato apyrase was used [3, 64, 117, 118]. Among several types of the potato apyrase, there is an isoform that preferentially hydrolyses ATP, with Km value of 8 µM and Kcat up to 2×103 µmole of product s-1 [129]. Thus, it appears to be a suitable tool for the NTPDase-based treatment. The Km values for ATP of human NTPDase1, NTPDase2, and NTPDase3 are 17, 70, and 75 μM, respectively, which suggests that NTPDase1 is the enzyme with the highest affinity towards ATP [9]. Other well characterized ecto-NTPDases are mammalian NTPDase1 and 2 from porcine brain cortex synaptosomes. NTPDase1 has Km value of 90 µM with respect to ATP as a substrate; however, the enzyme hydrolyses ATP and ADP equally well. On the contrary, NTPDase2 has a three times higher Km value (270 µM) in comparison to NTPDase1, but it prefers to hydrolyse ATP. Additionally, NTPDase1 and NTPDase2 from the porcine brain exhibit similar molecular activity (860 and 833 µmole of product × s-1, respectively) [130]. According to the above mentioned parameters, NTPDase1 activity could be involved in decreasing pathological ATP and ADP concentrations while NTPDase2 activity may contribute to lowering excessive amounts of ATP to physiological level while simultaneously increasing the concentration of another signalling molecule, ADP.

In the case of potential enzyme therapy, in addition to the selection of a suitable enzyme source, it is also necessary to provide an effective method of its delivery. Considering the ubiquitous presence of all the purinergic players in different cell types, tissues, and organs, the enhanced CNS tissue-targeted delivery and local action of purinergic molecules appears to be the best solution in CNS therapies. Similarly, NTPDase-based therapy aimed at decreasing ATP concentration in the nervous system requires a local delivery of catalytically active enzyme. Blood-brain barrier (BBB) and blood–cerebrospinal fluid barrier (BCSFB) permeation is a limiting factor for the delivery of such therapeutic agents. Furthermore, under pathological conditions, the blood-brain barrier often becomes dysfunctional. For testing CNS drug delivery, several in vitro models provide a reliable prediction of drug penetration including large molecules and artificial constructs. A current detailed review on the number of novel methods and models for BBB permeation has recently been published [131].

Biological approaches for potential NTPDase delivery to CNS can be based on cell penetrating peptides (CPPs) or colloidal drug carriers such as nanoparticles (NPs), micelles, liposomes, emulsions, and dendrimers. Many of these carriers can be effectively transported across various in vitro and in vivo BBB models by endocytosis and/or transcytosis [132, 133]. Decorating the surfaces of these colloidal particles with transferrin, glucose, or other receptor-specific ligands or antibodies may enhance their brain distribution. Biocompatibility, stability in circulation, and lack of immunogenicity are some other important factors to be considered [134]. Of great importance is the stability of enzyme immobilized on carrier particles, or encapsulated within them. NPs offer more stability to encapsulated molecules in biological fluids and protect them against

metabolic degradation, as compared to other colloidal systems such as liposomes or micelles [135]. The variability of their release mechanisms and their short-term release kinetics, are the key limitations of therapeutic enzyme delivery systems. The optimal carrier should release the entrapped enzymatic protein in a sustained manner for several weeks [134]. Therefore, the availability of effective brain-targeting drug delivery technology is one of the most significant challenges facing potential NTPDase-based treatment of CNS disorders. Recent advances in nanotechnology have been providing promising solutions to this challenge.

4.3. Stem cell-based Therapies

The application of stem cells as cellular vehicles for the delivery of therapeutic molecules has emerged within the last decade as an experimental approach to the therapy of nervous system disorders. In this regard, adult stem cells including neural stem and progenitor cells (NSC/NPC) and mesenchymal stem cells (MSC), as well as induced pluripotent stem cells (iPSC) can be promising candidates [136]. These stem cells exhibit a high tropism for disease-affected tissue via secreted growth factors and cytokines, and are easily integrated into the nervous tissue. Although various mammalian NSC/NPC and MSC have exhibited ecto-NTPDase activity on their surface [137-139], cells engineered to express therapeutic enzymes seems to be the most potent strategy. Stem cells genetically modified with NTPDase gene, and brain-targeted could effectively combine the high enzymatic activity with the local delivery to maintain the decrease in the ATP concentration.

The existence of multipotent neural stem cells (NSC) has been described in developing or adult mammalian CNS, including humans. However, in view of clinical tests, neural stem/progenitor cells have limited applications due to their hampered isolation and immunologic incompatibility. The use of autologous NSC is generally impossible, whereas allogeneic NSC may lead to problems regarding availability of cells from donors or their immunogenicity. Stable clonal lines of human NSC have recently been generated and utilized in stem-cell based therapy in animal models of human neurological disorders [140]. Moreover, clinical trial using human immortalized NPC as vehicles for the delivery of cytosine deaminase has recently been launched [136].

On the contrary, mesenchymal stem cells (MSC) can easily be derived from various adult tissues, such as bone marrow, the umbilical cord and adipose tissue. They can easily be expanded in culture and are amenable to genetic modification. Moreover, MSC show extraordinary immunomodulatory properties by suppressing proinflammatory cytokines production, and therefore, enable autologous and heterologous transplantation without the need of immunosuppression [141-143]. These qualities raise the clinical applicability of MSC engineered to express the therapeutic gene when dealing with the pathologies of CNS. In their study, Ryu and collaborators [144] evaluated the therapeutic effects for CNS disorders using human bone marrow-derived mesenchymal stem cells, engineered to secrete IFN-β, as delivery vehicles. MSC injected intravenously (i.v.) migrate preferentially to the sites of inflammation and may therefore be used for tissue directed immunosuppression, or for delivery of


therapeutic molecules to lesion site, following CNS injury. It seems that genetically manipulated MSC provide an attractive platform with lesion-targeting capability for the sustained production of therapeutic proteins in vivo. Progress along those cell lines has been made in the rodent models of neurodegenerative disorders and ischemic stroke, as well as in lysosomal storage disorders [described in 141, 145].

The discovery of human fibroblast-derived iPS cells [146] endorsed the speculation that stem cell therapies of the future may use the patient's own skin to obtain stem cells for the autologous cell therapy. The reprogrammed cells are patient-specific, so there is little need for immunosuppression. They can be pre-differentiated to neural progenitor cells and subjected to genetic modifications [147]. Improvements in the nuclear reprogramming techniques have made it safer, and more efficient. The original methods carried a significant risk of tumorigenesis due to the potential genomic integration of transgenes (oncogenes). However, it is now possible to induce pluripotent cells using recombinant proteins with membrane-permeable peptides or episomal plasmids, which are not integrated into the genome [148, 149]. Recent progressive studies have attempted to directly convert fibroblasts into induced neural progenitor cells (iNPCs) or functional neurons known as induced neurons (iNs), thus avoiding the production of a pluripotent intermediate state and circumventing the danger associated with tumorigenesis [149].

The application of engineered stem cells as carrier vehicles is a promising strategy for efficient enzyme delivery to CNS tissues. Although most of these approaches still remain in the experimental stages, continuing effort in developing new CNS delivery strategies will enable faster and widespread adoption of these techniques to clinical applications.

5. CONCLUDING REMARKS

It is commonly known that the neurological disorders effect from altered purinergic mechanisms, and the increased concentration of ATP underlies many diseases of the central nervous system. ATP, released locally in the brain tissue, acts within minutes as an excitotoxic molecule and at a longer time-scale it induces neuroinflammatory responses. These two effects often sum up, mainly due to the multiple purinergic receptors, activation of which leads to pleiotropic effects [67]. According to Marchetti and Abbracchio [150], a potential therapy for reduction of inflammation in neurodegenerative diseases should be based on the following approaches:

• Restoration of natural function of the brain to regulate inflammation,

• Development of combination therapies that target many factors which contribute to inflammation,

• Avoidance of systemic infections, as a correlation between the pre-existing peripheral inflammation, and the increased neuronal vulnerability has been demonstrated.

In our opinion, “purinergic drugs” fulfill all the above mentioned conditions. The first drugs that used the

pharmacological properties of purines were adenosine, and blockers of its re-uptake, i.e. dipyridamole. Another possible mechanism to increase the concentration of adenosine outside the cell is activation of ecto-5’-nucleotidase. Since adenosine itself, or its stable analogue N6-(L-2-phenylisopropyl)-adenosine are widely used as drugs in cardiovascular diseases or neuropathic pain, there is no urgent need to enzymatically regulate adenosine concentration.

The aspect of purinergic drugs also represents a promising field for the investigation of new therapies of the nervous system disorders. Current therapeutic strategies for CNS focus mainly on the use of P1 and P2 receptor antagonists or on the identification of potent and selective inhibitors for each member of the ecto-nucleotidase family. It is important to note that most of the analyzed receptor antagonists partially inhibit nucleotidase activity, so they indirectly participate in maintaining high ATP concentration. We postulate that up-regulation of ATP metabolism in the local environment of cells through ecto-NTPDase activation or delivery may provide the best therapeutic solution. The NTPDase-based strategies discussed here still remain in experimental stages but they do represent a promising start for the development of novel therapies of the central nervous system disorders.

LIST OF ABBREVIATIONS

AK = Adenylate kinase

AP = Alkaline phosphatase

BBB = Blood brain barrier

CNS = Central nervous system

CPP = Cell penetrating peptide

IL = Interleukin

iPSC = Induced pluripotent stem cell

LPS = Lipopolysaccharide

MSC = Mesenchymal stem cell

NP = Nanoparticle

NPP = Nucleotide pyrophosphohydrolase/ phosphodiesterase

NTPDase = Nucleoside triphosphate diphosphohydrolase

NSC = Neural stem cell

5’NT = 5’-nucleotidase

TNAP = Tissue nonspecific alkaline phosphatase.

CONFLICT OF INTEREST

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGEMENTS

Declared none.


SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher’s web site along with the published article.

REFERENCES [1] Burnstock G. Purinergic signaling and disorders of the central

nervous system. Nat. Rev. Drug Discov., 2008, 7(7), 575-590. [2] Bernier LP, Ase AR, Boué-Grabot É, Séguéla P. Inhibition of

P2X4 function by P2Y6 UDP receptors in microglia. Glia, 2013, 61(12), 2038-2049.

[3] Neher JJ, Neniskyte U, Hornik T, Brown GC. Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo. Glia, 2014, 62(9), 1463-1475.

[4] Hochhauser E, Cohen R, Waldman M, Maksin A, Isak A, Aravot D, Jayasekara PS, Müller CE, Jacobson KA, Shainberg A. P2Y2 receptor agonist with enhanced stability protects the heart from ischemic damage in vitro and in vivo. Purinergic Signal., 2013, 9, 633-642.

[5] Ralevic V, Burnstock G. Receptors for purines and pyrimidines. Pharmacol. Rev., 1998, 50, 413-492.

[6] Burnstock G, Verkhratsky A. Long-term (trophic) purinergic signaling: purinoceptors control cell proliferation, differentiation and death. Cell Death and Disease, 2010, 1, 1-10.

[7] Burnstock G. Purinergic signalling, past, present and future. Braz. J. Med. Biol., 2009, 42, 3-8.

[8] Robson SC, Sevigny J, Zimmermann H. The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance. Purinergic Signal., 2006, 2, 409-430.

[9] Zimmermann H, Zebisch M, Strater N. Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal., 2012, 8, 437-502.

[10] Bonan CD. Ectonucleotidases and nucleotide/nucleoside transporters as pharmacological targets for neurological disorders. CNS Neurol. Disord. Drug Targets, 2012, 11, 739-750.

[11] Zimmermann H, Braun N, Heine P, Kohring K, Marxen M, Sévigny J, Robson SC. The molecular and functional properties of E-NTPDase1, E-NTPDase2 and ecto-5'-nucleotidase in nervous tissue. In: Ecto-ATPases and related ectonucleotidases. Ed. by VanDuffel, L. and Lemmens, R. Shaker Publishing BV, Maastricht, 2000; pp. 9-20.

[12] Lazarowski E, Sesma J, Seminario-Vidal L, Kreda S. Molecular mechanism of purine and pyrimidyne nucleotide release. In: Pharmacology of Purine and Pyrimidine Receptors. Ed. by Jacobson K, Linden J, Academic Press, 2011, 221-261.

[13] Burnstock G, Volonté C. Pharmacology and therapeutic activity of purinergic drugs for disorders of the nervous system. CNS Neurol. Disord. Drug Targets, 2012, 11, 649-651.

[14] Bulavina L, Szulzewsky F, Rocha A, Krabbe G, Robson SC, Matyash V, Kettenmann H. NTPDase1 activity attenuates microglial phagocytosis. Purinergic Signal., 2013, 9(2), 199-205.

[15] Corti F, Olson KE, Marcus AJ, Levi R. The expression level of ecto-NTP diphosphohydrolase1/CD39 modulates exocytotic and ischemic release of neurotransmitters in a cellular model of sympathetic neurons. J. Pharmacol. Exp. Ther., 2011, 337(2), 524-532.

[16] Burnstock G. Purinergic signaling: pathophysiology and therapeutic potential. Keio J. Med., 2013, 62(3), 63-73.

[17] Anderson CM, Bergher JP, Swanson RA. ATP-induced ATP release from astrocytes. J. Neurochem., 2004, 88(1), 246-256.

[18] Dou Y, Wu H, Li H, Qin S, Wang Y, Li J, Lou H, Chen Z, Li X, Luo Q, Duan S. Microglial migration mediated by ATP-induced ATP release from lysosomes. Cell Res., 2012, 22(6), 1022-1033.

[19] Lohr C, Grosche A, Reichenbach A, Hirnet D. Purinergic neuron-glia interactions in sensory systems. Pflugers Arch., 2014, 466(10), 1859-1872.

[20] Yegutkin GG. Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade. Biochim. Biophys. Acta, 2008, 1783(5), 673-694.

[21] Kovács Z, Dobolyi A, Kékesi KA, Juhász G. 5'-nucleotidases, nucleosides and their distribution in the brain: pathological and therapeutic implications. Curr. Med. Chem., 2013, 20(34), 4217-4240.

[22] Basheer R, Strecker RE, Thakkar MM, McCarley RW. Adenosine and sleep-wake regulation. Prog. Neurobiol., 2004, 73(6), 379-396.

[23] Burnstock G. Kennedy C. Is there a basis for distinguishing two types of P2-purinoceptor? Gen. Pharmacol., 1985, 16, 433-440.

[24] Burnstock G. Purine and pyrimidine receptors. Cell. Mol. Life Sci., 2007, 64(12), 1471-1483.

[25] Burnstock G. Physiology and pathophysiology of purinergic neurotransmission. Physiol. Rev., 2007, 87(2), 659-797.

[26] Del Puerto A, Wandosell F, Garrido JJ. Neuronal and glial purinergic receptors functions in neuron development and brain disease. Front. Cell. Neurosci., 2013, 7, 197.

[27] Fields RD, Stevens B. ATP: an extracellular signaling molecule between neurons and glia. Trends Neurosci., 2003, 625-633.

[28] Ferrari D, Pizzirani C, Adinolfi E, Lemoli RM, Curti A, Idzko M, Panther E, Di Virgilio F. The P2X7 receptor: a key player in IL-1 processing and release. J. Immunol., 2006, 176, 3877-3883.

[29] Sperlágh B, Vizi ES, Wirkner K, Illes P. P2X7 receptors in the nervous system. Prog. Neurobiol., 2006, 78(6), 327-346.

[30] Zimmermann H, Braun N. Ecto-nucleotidases: Molecular structures, catalytic properties, and functional roles in the nervous system. Prog. Brain Res., 1999, 120, 371-385.

[31] Kukulski F, Lévesque SA, Lavoie EG, Lecka J, Bigonnesse F, Knowles AF, Robson SC, Kirley TL, Sévigny J. Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8. Purinergic Signal., 2005, 1(2), 193-204.

[32] Braun N, Sévigny J, Robson SC, Enjyoji K, Guckelberger O, Hammer K, Di Virgilio F, Zimmermann H. Assignment of ecto-nucleoside triphosphate diphosphohydrolase-1/cd39 expression to microglia and vasculature of the brain. Eur. J. Neurosci., 2000, 12(12), 4357-4366.

[33] Braun N, Sévigny J, Mishra SK, Robson SC, Barth SW, Gerstberger R, Hammer K, Zimmermann H. Expression of the ecto-ATPase NTPDase2 in the germinal zones of the developing and adult rat brain. Eur. J. Neurosci., 2003, 17(7), 1355-1364.

[34] Robson SC, Wu Y, Sun XF, Knosalla CK, Dwyer K, Enjyoji K. Ectonucleotidases of CD39 family modulate vascular inflammation m and thrombosis in transplantation. Semin. Thromb. Hemost., 2005, 31, 217-233.

[35] Wink MR, Braganhol E, Tamajusuku AS, Lenz G, Zerbini LF, Libermann TA, Sévigny J, Battastini AM, Robson SC. Nucleoside triphosphate diphosphohydrolase-2 (NTPDase2/CD39L1) is the dominant ectonucleotidase expressed by rat astrocytes. Neuroscience, 2006, 138, 421-432.

[36] Vorhoff T, Zimmermann H, Pelletier J, Sévigny J, Braun N. Cloning and characterization of the ecto-nucleotidase NTPDase3 from rat brain: Predicted secondary structure and relation to other members of the E-NTPDase family and actin. Purinergic Signal., 2005, 1, 259-270.

[37] Sakagami H, Aoki J, Natori Y, Nishikawa K, Kakehi Y, Natori Y, Arai H. Biochemical and molecular characterization of a novel choline-specific glycerophosphodiester phosphodiesterase belonging to the nucleotide pyrophosphatase/phosphodiesterase family. J. Biol. Chem., 2005, 280, 23084-23093.

[38] Tokumura A, Majima E, Kariya Y, Tominaga K, Kogure K, Yasuda K, Fukuzawa K. Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase. J. Biol. Chem., 2002, 277, 39436-39442.

[39] Cognato Gde P, Czepielewski R.S, Sarkis J.J, Bogo M.R Bonan CD. Expression mapping of ectonucleotide pyrophosphatase/ phosphodiesterase 1-3 (E-NPP1-3) in different brain structures during rat development. Int. J. Dev. Neurosci., 2008, 26(6), 593-598.

[40] Langer D, Hammer K, Koszalka P, Schrader J, Robson S, Zimmermann H. Distribution of ectonucleotidases in the rodent brain revisited. Cell Tissue Res., 2008, 334, 199-217.

[41] Kukulski F, Sévigny J, Komoszyński M. Comparative hydrolysis of extracellular adenine nucleotides and adenosine in synaptic membranes from porcine brain cortex, hippocampus, cerebellum and medulla oblongata. Brain Res., 2004, 1030, 49-56.

[42] Cunha R.A, Almeida T, Ribeiro J.A. Parallel modification of adenosine extracellular metabolism and modulatory action in the hippocampus of aged rats. J. Neurochem., 2001, 76(2), 372-382.

[43] Liu R, Xu H, Wei Z, Wang Y, Lin Y, Gong W. Crystal structure of human adenylate kinase 4 (L171P) suggests the role of hinge


region in protein domain motion. Biochem. Biophys. Res. Commun., 2009, 379, 92-97.

[44] Dzeja P, Terzic A. Adenylate kinase and AMP signaling networks: metabolic monitoring, signal communication and body energy sensing. Int. J. Mol. Sci., 2009, 10, 1729-1772.

[45] Stanojevic V, Habener JF, Holz GG, Leech CA. Cytosolic adenylate kinases regulate K-ATP channel activity in human beta-cells. Biochem. Biophys. Res. Commun., 2008, 368, 614-619.

[46] Carrasco AJ, Dzeja PP, Alekseev AE, Pucar D, Zingman LV, Abraham MR, Hodgson D, Bienengraeber M, Puceat M, Janssen E, Wieringa B, Terzic A. Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels. Proc. Natl. Acad. Sci. USA, 2001, 98, 7623-7628.

[47] Donaldson SH, Picher M, Boucher RC. Secreted and cell-associated adenylate kinase and nucleoside diphosphokinase contribute to extracellular nucleotide metabolism on human airway surfaces. Am. J. Respir. Cell Mol. Biol., 2002, 26, 209-215.

[48] Quillen EE, Haslam GC, Samra HS, Amani-Taleshi D, Knight JA, Wyatt DE, Bishop SC, Colvert KK, Richter ML, Kitos PA. Ectoadenylate kinase and plasma membrane ATP synthase activities of human vascular endothelial cells. J. Biol. Chem., 2006, 281, 20728-20737.

[49] Sperlágh B, Vizi ES. Extracellular interconversion of nucleotides reveals an ecto-adenylate kinase activity in the rat hippocampus. Neurochem Res., 2007, 32, 1978-1989.

[50] Noma T, Yoon YS, Nakazawa A. Overexpression of NeuroD in PC12 cells alters morphology and enhances expression of the adenylate kinase isozyme 1 gene. Brain Res. Mol. Brain Res., 1999, 67, 53-63.

[51] Nagy AK, Houser CR, Delgado-Escueta AV. Synaptosomal ATPase activities in temporal cortex and hippocampal formation of humans with focal epilepsy. Brain Res., 1990, 529, 192-201.

[52] Neary JT, Zimmermann H. Trophic functions of nucleotides in the central nervous system. Trends Neurosci., 2009, 32(4), 189-198.

[53] Teppema LJ, Dahan A. The ventilatory response to hypoxia in mammals: mechanisms, measurement, and analysis. Physiol. Rev., 2010, 90, 675-754.

[54] Wardas J. Neuroprotective role of adenosine in the CNS. Pol. J. Pharmacol., 2002, 54(4), 313-326.

[55] Domercq M, Vázquez-Villoldo N, Matute C. Neurotransmitter signaling in the pathophysiology of microglia. Front. Cell. Neurosci., 2013, 19, 7-49.

[56] Domercq M, Perez-Samartin A, Aparicio D, Alberdi E, Pampliega O, Matute C. P2X7 receptors mediate ischemic damage to oligodendrocytes. Glia, 2010, 58(6), 730-740.

[57] Wixey JA, Reinebrant HE, Carty ML, Buller KM. Delayed P2X4R expression after hypoxia-ischemia is associated with microglia in the immature rat brain. J. Neuroimmunol., 2009, 212(1-2), 35-43.

[58] Colgan SP, Eltzschig HK, Eckle T, Thompson LF. Physiological roles for ecto-5'-nucleotidase (CD73). Purinergic Signal., 2006, 2(2), 351-360.

[59] Lopes LV, Sebastião AM, Ribeiro JA. Adenosine and related drugs in brain diseases, present and future in clinical trials. Curr. Top. Med. Chem., 2011, 11(8), 1087-1101.

[60] Boison D. Modulators of nucleoside metabolism in the therapy of brain diseases. Curr. Top. Med. Chem. 2011, 11(8), 1068-1086.

[61] Bours MJ, Swennen EL, Di Virgilio F, Cronstein BN, Dagnelie PC. Adenosine 5'-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol. Ther., 2006, 112(2), 358-404.

[62] Idzko M, Hammad H, van Nimwegen M, Kool M, Willart MA, Muskens F, Hoogsteden HC, Luttmann W, Ferrari D, Di Virgilio F, Virchow JC Jr, Lambrecht BN. Extracellular ATP triggers and maintains asthmatic airway inflammation by activating dendritic cells. Nat. Med., 2007, 13(8), 913-919.

[63] Idzko M, Ferrari D, Eltzschig HK. Nucleotide signalling during inflammation. Nature, 2014, 509, 310-317.

[64] Amaral SS, Oliveira AG, Marques PE, Quintão JL, Pires DA, Resende RR, Sousa BR, Melgaço JG, Pinto MA, Russo RC, Gomes AK, Andrade LM, Zanin RF, Pereira RV, Bonorino C, Soriani FM, Lima CX, Cara DC, Teixeira MM, Leite MF, Menezes GB. Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity. Cell Commun. Signal., 2013, 11(1), 10.

[65] Friedle SA, Curet MA, Watters JJ. Recent patents on novel P2X7 receptor antagonists and their potential for reducing central nervous

system inflammation. Recent Pat. CNS Drug Discov., 2010, 5(1), 35-45.

[66] Di Virgilio F, Ceruti S, Bramanti P, Abbracchio MP. Purinergic signalling in inflammation of the central nervous system. Trends Neurosci., 2009, 32(2),79-87.

[67] Franke H, Illes P. Nucleotide signaling in astrogliosis. Neurosci. Lett., 2014, 565, 14-22.

[68] Ogata T, Chuaia M, Morinoa T, Yamamotoa H, Nakamura Y, Schubert P. Adenosine triphosphate inhibits cytokine release from lipopolysaccharide-activated microglia via P2y receptors. Brain Res., 2003, 981, 174-183.

[69] Seo DR, Kim KY, Lee YB. Interleukin-10 expression in lipopolysaccharide-activated microglia is mediated by extracellular ATP in an autocrine fashion. Neuroreport, 2004, 15(7), 1157-1161.

[70] Swennen EL, Bast A, Dagnelie PC. Immunoregulatory effects of adenosine 5'-triphosphate on cytokine release from stimulated whole blood. Eur. J. Immunol., 2005, 35(3), 852-858.

[71] Swennen EL, Bast A, Dagnelie PC. Purinergic receptors involved in the immunomodulatory effects of ATP in human blood. Biochem. Biophys. Res. Commun., 2006, 348(3), 1194-1199.

[72] Kucher BM, Neary JT. Bi-functional effects of ATP/P2 receptor activation on tumor necrosis factor-alpha release in lipopolysaccharide-stimulated astrocytes. J. Neurochem., 2005, 92(3), 525-535.

[73] Skaper SD, Giusti P, Facci L. Microglia and mast cells: two tracks on the road to neuroinflammation. FASEB J., 2012, 26(8), 3103-3117.

[74] Augusto E, Matos M, Sévigny J, El-Tayeb A, Bynoe MS, Müller CE, Cunha RA, Chen JF. Ecto-5'-nucleotidase (CD73)-mediated formation of adenosine is critical for the striatal adenosine A2A receptor functions. J. Neurosci., 2013, 33(28), 11390-11399.

[75] Goepfert C, Sundberg C, Sévigny J, Enjyoji K, Hoshi T, Csizmadia E, Robson S. Disordered cellular migration and angiogenesis in cd39-null mice. Circulation, 2001, 104(25), 3109-3115.

[76] Mizumoto N, Kumamoto T, Robson SC, Sévigny J, Matsue H, Enjyoji K, Takashima A. CD39 is the dominant Langerhans cell-associated ecto-NTPDase: modulatory roles in inflammation and immune responsiveness. Nat. Med., 2002, 8(4), 358-365.

[77] Iandiev I, Wurm A, Pannicke T, Wiedemann P, Reichenbach A, Robson SC, Zimmermann H, Bringmann A. Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling. Purinergic Signal., 2007, 3(4), 423-433.

[78] Kauffenstein G, Fürstenau CR, D'Orléans-Juste P, Sévigny J. The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation. Br. J. Pharmacol., 2010, 159(3), 576-585.

[79] Koszalka P, Ozüyaman B, Huo Y, Zernecke A, Flögel U, Braun N, Buchheiser A, Decking UK, Smith ML, Sévigny J, Gear A, Weber AA, Molojavyi A, Ding Z, Weber C, Ley K, Zimmermann H, Gödecke A, Schrader J. Targeted disruption of cd73/ecto-5'-nucleotidase alters thromboregulation and augments vascular inflammatory response. Circ. Res., 2004, 95(8), 814-821.

[80] Gao HM, Hong JS. Why neurodegenerative diseases are progressive: uncontrolled inflammation drives disease progression. Trends Immunol., 2008, 29(8), 357-365.

[81] Tansey MG, Goldberg MS. Neuroinflammation in Parkinson's disease: its role in neuronal death and implications for therapeutic intervention. Neurobiol. Dis., 2010, 37(3), 510-518.

[82] Heneka MT, Rodríguez JJ, Verkhratsky A. Neuroglia in neurodegeneration. Brain Research Rev., 2010, 63, 189-211.

[83] Heine C, Wegner A, Grosche J, Allgaier C, Illes P, Franke H. P2 receptor expression in the dopaminergic system of the rat brain during development. Neuroscience, 2007, 149(1), 165-181.

[84] Krügel U, Kittner H, Franke H, Illes P. Stimulation of P2 receptors in the ventral tegmental area enhances dopaminergic mechanisms in vivo. Neuropharmacol., 2001, 40(8), 1084-1093.

[85] Trendelenburg AU, Bültmann R. P2 receptor-mediated inhibition of dopamine release in rat neostriatum. Neuroscience, 2000, 96(2), 249-252.

[86] Block ML, Hong JS. Chronic microglial activation and progressive dopaminergic neurotoxicity. Biochem. Soc. Trans., 2007, 35(Pt 5), 1127-1132.

[87] Amadio S, Montilli C, Picconi B, Calabresi P, Volonté C. Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study. Purinergic Signal., 2007, 3(4), 389-398.


[88] Able SL, Fish RL, Bye H, Booth L, Logan YR, Nathaniel C, Hayter P, Katugampola SD. Receptor localization, native tissue binding and ex vivo occupancy for centrally penetrant P2X7 antagonists in the rat. Br. J. Pharmacol., 2011, 162(2), 405-414.

[89] Carmo MR, Menezes AP, Nunes AC, Pliássova A, Rolo AP, Palmeira CM, Cunha RA, Canas PM, Andrade GM. The P2X7 receptor antagonist Brilliant Blue G attenuates contralateral rotations in a rat model of Parkinsonism through a combined control of synaptotoxicity, neurotoxicity and gliosis. Neuropharmacol., 2014, 81, 142-152.

[90] Choi YM, Jang JY, Jang M, Kim SH, Kang YK, Cho H, Chung S, Park MK. Modulation of firing activity by ATP in dopamine neurons of the rat substantia nigra pars compacta. Neuroscience, 2009, 160(3), 587-595.

[91] Hernández VS, Luquín S, Jáuregui-Huerta F, Corona-Morales AA, Medina MP, Ruíz-Velasco S, Zhang L. Dopamine receptor dysregulation in hippocampus of aged rats underlies chronic pulsatile L-Dopa treatment induced cognitive and emotional alterations. Neuropharmacol., 2014, 82, 88-100.

[92] Moore AH, Bigbee MJ, Boynton GE, Wakeham CM, Rosenheim HM, Staral CJ, Morrissey JL, Hund AK. Non-steroidal anti-inflammatory drugs in Alzheimer's disease and Parkinson's disease: reconsidering the role of neuroinflammation Pharmaceuticals, 2010, 3, 1812-1841.

[93] Akiyama H, Barger S, Barnum S, Bradt B, Bauer J, Cole GM, Cooper NR, Eikelenboom P, Emmerling M, Fiebich BL, Finch CE, Frautschy S, Griffin WS, Hampel H, Hull M, Landreth G, Lue L, Mrak R, Mackenzie IR, McGeer PL, O'Banion MK, Pachter J, Pasinetti G, Plata-Salaman C, Rogers J, Rydel R, Shen Y, Streit W, Strohmeyer R, Tooyoma I, Van Muiswinkel FL, Veerhuis R, Walker D, Webster S, Wegrzyniak B, Wenk G, Wyss-Coray T. Inflammation and Alzheimer's disease. Neurobiol. Aging, 2000, 21(3), 383-421.

[94] Kortekaas R, Leenders KL, van Oostrom JC, Vaalburg W, Bart J, Willemsen AT, Hendrikse NH. Bloodbrain barrier dysfunction in parkinsonian midbrain in vivo. Ann. Neurol., 2005, 57(2), 176-179.

[95] Brochard V, Combadiere B, Prigent A, Laouar Y, Perrin A, Beray-Berthat V, Bonduelle O, Alvarez- Fischer D, Callebert J, Launay JM, Duyckaerts C, Flavell RA, Hirsch EC, Hunot S. Infiltration of CD4+ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease. J. Clin. Invest., 2009, 119, 182–192.

[96] Nelson PT, Abner EL, Schmitt FA, Kryscio RJ, Jicha GA, Smith CD, Davis DG, Poduska JW, Patel E, Mendiondo MS, Markesbery WR. Modeling the association between 43 different clinical and pathological variables and the severity of cognitive impairment in a large autopsy cohort of elderly persons. Brain Pathol., 2010, 20(1), 66-79.

[97] Nelson PT, Alafuzoff I, Bigio EH, Bouras C, Braak H, Cairns NJ, Castellani RJ, Crain BJ, Davies P, Del Tredici K, Duyckaerts C, Frosch MP, Haroutunian V, Hof PR, Hulette CM, Hyman BT, Iwatsubo T, Jellinger KA, Jicha GA, Kövari E, Kukull WA, Leverenz JB, Love S, Mackenzie IR, Mann DM, Masliah E, McKee AC, Montine TJ, Morris JC, Schneider JA, Sonnen JA, Thal DR, Trojanowski JQ, Troncoso JC, Wisniewski T, Woltjer RL, Beach TG. Correlation of Alzheimer disease neuropathologic changes with cognitive status: a review of the literature. J. Neuropathol. Exp. Neurol., 2012, 71(5), 362-381.

[98] Shrivastava AN, Kowalewski JM, Renner M, Bousset L, Koulakoff A, Melki R, Giaume C, Triller A. β-amyloid and ATP-induced diffusional trapping of astrocyte and neuronal metabotropic glutamate type-5 receptors. Glia, 2013, 61(10), 1673-1686.

[99] McLarnon JG, Ryu JK, Walker DG, Choi HB. Upregulated expression of purinergic P2X(7) receptor in Alzheimer disease and amyloid-beta peptide-treated microglia and in peptide-injected rat hippocampus. J. Neuropathol. Exp. Neurol., 2006, 65(11), 1090-1097.

[100] Varma R, Chai Y, Troncoso J, Gu J, Xing H, Stojilkovic SS, Mattson MP, Haughey NJ. Amyloid-beta induces a caspase-mediated cleavage of P2X4 to promote purinotoxicity. Neuromolecular Med., 2009, 11(2), 63-75.

[101] Camden JM, Schrader AM, Camden RE, González FA, Erb L, Seye CI, Weisman GA. P2Y2 nucleotide receptors enhance alpha-secretase-dependent amyloid precursor protein processing. J. Biol. Chem., 2005, 280(19), 18696-18702.

[102] Gandelman M, Peluffo H, Beckman JS, Cassina P, Barbeito L. Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis. J. Neuroinflammation, 2010, 7, 33.

[103] Spanevello RM, Mazzanti CM, Kaizer R, Zanin R, Cargnelutti D, Hannel L, Côrrea M, Mazzanti A, Festugatto R, Graça D, Schetinger MR, Morsch VM. Apyrase and 5'-nucleotidase activities in synaptosomes from the cerebral cortex of rats experimentally demyelinated with ethidium bromide and treated with interferon-beta. Neurochem. Res., 2006, 31(4), 455-462.

[104] Burnstock G. Purinergic mechanisms and pain - an update. Eur. J. Pharmacol., 2013, 716(1-3), 24-40.

[105] Chen CC, Akopian AN, Sivilotti L, Colquhoun D, Burnstock G, Wood JN. A P2X purinoceptor expressed by a subset of sensory neurons. Nature, 1995, 377(6548), 428-431.

[106] Kuroda H, Shibukawa Y, Soya M, Masamura A, Kasahara M, Tazaki M, Ichinohe T. Expression of P2X� and P2X� receptors in rat trigeminal ganglion neurons. Neuroreport, 2012, 23(13), 752-756.

[107] Czarnecka J, Cieślak M, Komoszyński M. Application of solid phase extraction and high-performance liquid chromatography to qualitative and quantitative analysis of nucleotides and nucleosides in human cerebrospinal fluid. J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci., 2005, 822(1-2), 85-90.

[108] Kettenmann H, Hanisch UK, Noda M, Verkhratsky A. Physiology of microglia. Physiol. Rev., 2011, 91(2), 461-553.

[109] Henshall DC, Diaz-Hernandez M, Miras-Portugal MT, Engel T. P2X receptors as targets for the treatment of status epilepticus. Front. Cell. Neurosci., 2013, 7, 237.

[110] Chang BS, Lowenstein DH. Epilepsy. N. Engl. J. Med., 2003, 349(13), 1257-1266.

[111] Ottman R, Risch N, Hauser WA, Pedley TA, Lee JH, Barker-Cummings C, Lustenberger A, Nagle KJ, Lee KS, Scheuer ML. Localization of a gene for partial epilepsy to chromosome 10q. Nat. Genet., 1995, 10(1), 56-60.

[112] Zgodziński W, Rubaj A, Kleinrok Z, Sieklucka-Dziuba M., Effect of adenosine A1 and A2 receptor stimulation on hypoxia-induced convulsions in adult mice. Pol. J. Pharmacol., 2001, 53(1), 83-92.

[113] Klaft ZJ, Schulz SB, Maslarova A, Gabriel S, Heinemann U, Gerevich Z. Extracellular ATP differentially affects epileptiform activity via purinergic P2X7 and adenosine A1 receptors in naive and chronic epileptic rats. Epilepsia, 2012, 53(11), 1978-1986.

[114] Bonan CD, Walz R, Pereira GS, Worm PV, Battastini AM, Cavalheiro EA, Izquierdo I, Sarkis JJ. Changes in synaptosomal ecto-nucleotidase activities in two rat models of temporal lobe epilepsy. Epilepsy Res., 2000, 39(3), 229-238.

[115] Al-Rashida M, Iqbal J. Therapeutic potentials of ecto-nucleoside triphosphate diphosphohydrolase, ecto-nucleotide pyrophosphatase/ phosphodiesterase, ecto-5'-nucleotidase, and alkaline phosphatase inhibitors. Med. Res. Rev., 2014, 34(4), 703-743.

[116] Brunschweiger A, Iqbal J, Umbach F, Scheiff AB, Munkonda MN, Sévigny J, Knowles AF, Müller CE. Selective nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) inhibitors: nucleotide mimetics derived from uridine-5'-carboxamide. J. Med. Chem., 2008, 51(15), 4518-4528.

[117] Bayliss J, Delarosa S, Wu J, Peterson JR, Eboda ON, Su GL, Hemmila M, Krebsbach PH, Cederna PS, Wang SC, Xi C, Levi B. Adenosine triphosphate hydrolysis reduces neutrophil infiltration and necrosis in partial-thickness scald burns in mice. J. Burn Care Res., 2014, 35(1), 54-61.

[118] Li P, Cao J, Chen Y, Wang W, Yang J. Apyrase protects against allergic airway inflammation by decreasing the chemotactic migration of dendritic cells in mice. Int. J. Mol. Med., 2014, 34(1), 269-275.

[119] Pinsky DJ, Broekman MJ, Peschon JJ, Stocking KL, Fujita T, Ramasamy R, Connolly ES Jr, Huang J, Kiss S, Zhang Y, Choudhri TF, McTaggart RA, Liao H, Drosopoulos JH, Price VL, Marcus AJ, Maliszewski CR. Elucidation of the thromboregulatory role of CD39/ectoapyrase in the ischemic brain. J. Clin. Invest., 2002, 109(8), 1031-1040.

[120] Marcus AJ, Broekman MJ, Drosopoulos JH, Islam N, Pinsky DJ, Sesti C, Levi R. Metabolic control of excessive extracellular nucleotide accumulation by CD39/ecto-nucleotidase-1: implications for ischemic vascular diseases. J. Pharmacol. Exp. Ther., 2003, 305(1), 9-16.


[121] Moeckel D, Jeong SS, Sun X, Broekman MJ, Nguyen A, Drosopoulos JH, Marcus AJ, Robson SC, Chen R, Abendschein D.Optimizing human apyrase to treat arterial thrombosis and limit reperfusion injury without increasing bleeding risk. Sci. Transl. Med., 2014, 6(248), 248ra105.

[122] Schetinger MR, Morsch VM, Bonan CD, Wyse AT. NTPDase and 5'-nucleotidase activities in physiological and disease conditions: new perspectives for human health. Biofactors, 2007, 31(2), 77-98.

[123] Schmatz R, Mazzanti CM, Spanevello R, Stefanello N, Gutierres J, Maldonado PA, Corrêa M, da Rosa CS, Becker L, Bagatini M, Gonçalves JF, Jaques Jdos S, Schetinger MR, Morsch VM. Ecto-nucleotidase and acetylcholinesterase activities in synaptosomes from the cerebral cortex of streptozotocin-induced diabetic rats and treated with resveratrol. Brain Res. Bull., 2009, 80(6), 371-376.

[124] Wink MR, Lenz G, Rodnight R, Sarkis JJ, Battastini AM. Identification of brain ecto-apyrase as a phosphoprotein. Mol. Cell. Biochem., 2000, 213(1-2), 11-16.

[125] Wang CJ, Vlajkovic SM, Housley GD, Braun N, Zimmermann H, Robson SC, Sévigny J, Soeller C, Thone PR. C-terminal splicing of NTPDase2 provides distinctive catalytic properties, cellular distribution and enzyme regulation. Biochem. J., 2005, 385, 729-736.

[126] Baek AE, Kanthi Y, Sutton NR, Liao H, Pinsky DJ. Regulation of ecto-apyrase CD39 (ENTPD1) expression by phosphodiesterase III (PDE3). FASEB J., 2013, 27(11), 4419-4428.

[127] Zhong X, Buddha M, Guidotti G, Kriz R, Somers W, Mosyak L. Expression, purification and crystallization of the ecto-enzymatic domain of rat E-NTPDase1 CD39. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun., 2008, 64(Pt 11), 1063-1065.

[128] Kozakiewicz A, Neumann P, Banach M, Komoszyński M, Wojtczak A. Modeling studies of potato nucleoside triphosphate diphosphohydrolase NTPDase1: an insight into the catalytic mechanism. Acta Biochim. Pol., 2008, 55(1), 141-150.

[129] Kettlun AM, Espinosa V, García L, Valenzuela MA. Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility. Phytochemistry, 2005, 66(9), 975-982.

[130] Kukulski F, Komoszyński M. Purification and characterization of NTPDase1 (ecto-apyrase) and NTPDase2 (ecto-ATPase) from porcine brain cortex synaptosomes. Eur. J. Biochem., 2003, 270(16), 3447-3454.

[131] Abbott NJ. Blood-brain barrier structure and function and the challenges for CNS drug delivery. J. Inherit. Metab. Dis., 2013, 36(3), 437-449.

[132] Wong HL, Wu XY, Bendayan R. Nanotechnological advances for the delivery of CNS therapeutics. Adv. Drug Deliv. Rev., 2012, 64(7), 686-700.

[133] Domínguez A, Suárez-Merino B, Goñi-de-Cerio F. Nanoparticles and blood-brain barrier: the key to central nervous system diseases. J. Nanosci. Nanotechnol., 2014, 14(1), 766-779.

[134] Lu CT, Zhao YZ, Wong HL, Cai J, Peng L, Tian XQ. Current approaches to enhance CNS delivery of drugs across the brain barriers. Int. J. Nanomedicine, 2014, 9, 2241-2257.

[135] Kreuter J. Nanoparticulate systems for brain delivery of drugs. Adv. Drug Deliv. Rev., 2001, 47, 65-81.

[136] Tabatabai G, Wick W, Weller M. Stem cell-mediated gene therapies for malignant gliomas: a promising targeted therapeutic approach? Discov. Med., 2011, 11(61), 529-536.

[137] Roszek K, Błaszczak A, Wujak M, Komoszyński M. Nucleotides metabolizing ecto-enzymes as possible markers of mesenchymal stem cell osteogenic differentiation. Biochem. Cell Biol., 2013, 91(3), 176-181.

[138] Roszek K, Bomastek K, Drożdżal M, Komoszyński M. Dramatic differences in activity of purines metabolizing ecto-enzymes between mesenchymal stem cells isolated from human umbilical cord blood and umbilical cord tissue. Biochem. Cell Biol., 2013, 91(6), 519-525.

[139] Mishra SK, Braun N, Shukla V, Füllgrabe M, Schomerus C, Korf HW, Gachet C, Ikehara Y, Sévigny J, Robson SC, Zimmermann H. Extracellular nucleotide signaling in adult neural stem cells: synergism with growth factor-mediated cellular proliferation. Development, 2006, 133, 675-684.

[140] Kim SU. Genetically engineered human neural stem cells for brain repair in neurological diseases. Brain Dev., 2007, 29(4), 193-201.

[141] Azari MF, Mathias L, Ozturk E, Cram DS, Boyd RL, Petratos S. Mesenchymal stem cells for treatment of CNS injury. Curr. Neuropharmacol., 2010, 8(4), 316-323.

[142] De Miguel MP, Fuentes-Julián S, Blázquez-Martínez A, Pascual CY, Aller MA, Arias J, Arnalich-Montiel F. Immunosuppressive properties of mesenchymal stem cells: advances and applications. Curr. Mol. Med., 2012, 12(5), 574-591.

[143] Lotfinegad P, Shamsasenjan K, Movassaghpour A, Majidi J, Baradaran B. Immunomodulatory nature and site specific affinity of mesenchymal stem cells: a hope in cell therapy. Adv. Pharm. Bull., 2014, 4(1), 5-13.

[144] Ryu CH, Park KY, Hou Y, Jeong CH, Kim SM, Jeun SS. Gene therapy of multiple sclerosis using interferon β -secreting human bone marrow mesenchymal stem cells. Biomed. Res. Int., 2013, 2013, 696-738.

[145] Reiser J, Zhang XY, Hemenway CS, Mondal D, Pradhan L, La Russa VF. Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases. Expert Opin. Biol. Ther., 2005, 5(12), 1571-1584.

[146] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007, 131(5), 861-872.

[147] Okano H, Nakamura M, Yoshida K, Okada Y, Tsuji O, Nori S, Ikeda E, Yamanaka S, Miura K. Steps toward safe cell therapy using induced pluripotent stem cells. Circ. Res., 2013, 112(3), 523-533.

[148] Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuzdak G, Schöler HR, Duan L, Ding S. Generation of induced pluripotent stem cells using recombinant proteins. Cell Stem Cell, 2009, 4, 381-384.

[149] Ito D, Okano H, Suzuki N. Accelerating progress in induced pluripotent stem cell research for neurological diseases. Ann. Neurol., 2012, 72(2), 167-174.

[150] Marchetti B, Abbracchio MP. To be or not to be (inflamed) - is that the question in anti-inflammtory drug therapy of neurodegenerative disorders? Trends Pharmacol. Sci., 2005, 26, 517-525.

Received: July 11, 2014 Revised: November 26, 2014 Accepted: November 29, 2014

Is Ecto-nucleoside Triphosphate Diphosphohydrolase (NTPDase)-based Therapy of Central Nervous System...

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