CNS & Neurological Disorders - Drug Targets (Formerly Current Drug Targets - CNS & Neurological Disorders) - Volume 11, Issue 6, 2012

The role of A1 and A3 receptors is discussed based on data almost exclusively obtained in the hippocampus. This brain area, where A1 receptor expression predominates, has been a matter of intensive research in the adenosine field. Interestingly, in the last decade, the relevance of the much less expressed adenosine receptor in the hippocampus, the A2A receptor, has been put forward. These two high affinity receptors operate as effective regulators of a number of neurotransmitters and/or neuromodulators, through modulation of their release, action, or even inactivation. Therefore, A1 and A2A receptors constitute a must in the discussion about adenosine receptors in the hippocampus, and consequently, about the potential implications of their pharmacological manipulation and drug targeting.

Adenosine A2B and, much more importantly, adenosine A2A receptors modulate many physiological and pathological processes in the brain. In this review, the most recent evidence concerning the role of such receptors and their potential therapeutic relevance is discussed. The low affinity of A2B receptors for adenosine implies that they might represent a good therapeutic target, since they are activated only under pathological conditions (when adenosine levels raise up to micromolar concentrations). The availability of selective ligands for A2B receptors would allow exploration of such an hypothesis. Since adenosine A2A receptors mediate both potentially neuroprotective and potentially neurotoxic effects, their role in neurodegenerative diseases is highly controversial. Nevertheless, A2A receptor antagonists have shown clear antiparkinsonian effects, and a great interest exists on the role of A2A receptors in Alzheimer’s disease, brain ischaemia, spinal cord injury, drug addiction and other conditions. In order to establish whether such receptors represent a target for CNS diseases, at least two conditions are needed: the full comprehension of A2A-dependent mechanisms and the availability of ligands capable of discriminating among the different receptor populations.

P2X receptors are homo- or heterotrimeric ATP-gated cation channels that assemble from seven subunits, P2X1-P2X7. To our knowledge, no drug that acts on the P2X1 or P2X2 receptors in the CNS or elsewhere in the body has been approved, nor is there such a drug currently in clinical trials. Only a few non-drug-like antagonists such as the suramin derivatives NF449 and NF770 and the anthraquinone derivative PSB-1011 are available as pharmacological tools to block the P2X1 and P2X2 receptors, respectively. The focus of this review is twofold. First, we review the current knowledge of the role of the P2X1 and P2X2 receptors in normal and pathological CNS functions as inferred from experiments with wild-type, P2X1 knockout and P2X2 knockout mice. From the available data we conclude that the P2X1 and P2X2 receptors may have therapeutic potential as targets for neuroprotective drugs. Second, we review the impact of the recent resolution of the crystal structure of the zebrafish P2X4 receptor in the apo closed state and the ATP-bound open state. The P2X4 crystal structure opens the exciting possibility to generate P2X homology models for a rational drug design. In silico docking experiments with a homology-modeled rat P2X2 receptor revealed an almost perfect coordination of the nanomolar potent P2X2 antagonist NF770 through strong polar interactions between the acidic groups of NF770 and the mostly basic groups of the ATP-binding pocket. Such structural information might be helpful in designing drug-like compounds that function as selective P2X receptor antagonists without the pharmacokinetic limitations of the currently available antagonists.

The strong expression of ATP-gated P2X3 receptors by a subpopulation of sensory neurons indicates the important role of these membrane proteins in nociceptive signaling in health and disease, especially when the latter is accompanied by chronic pain syndromes. Molecular and cell biology studies have shown that these receptors exist mainly as trimeric homomers, and, in part, as heteromers (assembly of two P2X3 subunits with one P2X2). Recent investigations have suggested distinct molecular determinants responsible for agonist binding and channel opening for transmembrane flux of sodium, calcium and potassium ions. Trimeric P2X3 receptors are rapidly activated by ATP and can be strongly desensitized in the continuous presence of the agonist. Thus, the factors controlling the degree of desensitization and the time necessary to recover from it are essential elements to determine how efficiently and how often the P2X3 receptor can signal pain. Endogenous substances, widely thought to be involved in triggering pain especially in pathological conditions, can potently modulate the expression and function of P2X3 receptors, with differential changes in response amplitude, desensitization and recovery. Hence, studying P2X3 receptors can lead not only to the design of novel antagonists as analgesics, but also to identify intracellular interactors that may be targeted to downregulate P2X3 receptors. Strong facilitation of P2X3 receptor function is induced by endogenous substances like the neuropeptide calcitonin gene-related peptide and the neurotrophins nerve growth factor and brain-derived neurotrophic factor. These substances possess distinct mechanisms of action on P2X3 receptors, generally attributable to discrete phosphorylation of N- or C-terminal P2X3 domains.

We have learned various data on the role of purinoceptors (P2X4, P2X7, P2Y6 and P2Y12 receptors) expressed in spinal microglia and several factors that presumably activate microglia in neuropathic pain after peripheral nerve injury. Especially P2X4 receptors (P2X4Rs) make a critical contribution to the pain processing. P2X4Rs of microglia might be promising targets for treating neuropathic pain. A predicted therapeutic benefit of interfering with microglial P2X4Rs may be that normal pain sensitivity would be unaffected since expression or activity of most of these receptors are upregulated or enhanced predominantly in activated microglia in the spinal cord where damaged sensory fibers project. Recently, we found that CCL21 regulates the expression of P2X4Rs in different manners, respectively. These new findings also provide novel targets for developing anti-neuropathic pain medicines.

Purine nucleotides are well established as extracellular signaling molecules. P2X7 receptors (P2X7Rs) are members of the family of ionotropic ATP-gated receptors. Their activity can be found in a limited number of cell types, but is readily detectable in cells of hemopoietic lineage including macrophages, microglia, and certain lymphocytes, and mediates the influx of Ca2+ and Na+ as well as the release of pro-inflammatory cytokines. Amongst P2X receptors, P2X7Rs behave as a bifunctional molecule. The binding of ATP induces within milliseconds the opening of a channel selective for small cations, and within seconds a larger pore opens which allows permeation by molecules with a mass of up to 900 Da. In humans at least, the P2RX7 gene is highly polymorphic, and genetic differences within P2X7R affect receptor pore formation and channel function. ATP can act as a neurotransmitter, while the presence of P2X7Rs on immune cells suggests that they also regulate immune function and inflammatory responses. In addition, activation of the P2X7R has dramatic cytotoxic properties. The role of extracellular ATP and purinoceptors in cytokine regulation and neurological disorders is, in fact, the focus of a rapidly expanding area of research. P2X7Rs may affect neuronal cell death by regulating the processing and release of interleukin-1β, a key mediator in neurodegeneration, chronic inflammation, and chronic pain. Activation of P2X7Rs provides an inflammatory stimulus, and P2X7R-deficient mice display a marked attenuation of inflammatory responses, including models of neuropathic and chronic inflammatory pain. Moreover, P2X7R activity, by regulating the release of pro-inflammatory cytokines, may be involved in the pathophysiology of neuropsychiatric disorders. The P2X7R may thus represent a critical communication link between the nervous and immune systems, while providing a target for therapeutic exploitation. In this review we discuss current biology and pharmacology of the P2X7R, as well as insights into the role for this receptor in neurological/psychiatric diseases.

P2Y receptors for extracellular nucleotides are coupled to activation of a variety of G proteins and stimulate diverse intracellular signaling pathways that regulate functions of cell types that comprise the central nervous system (CNS). There are 8 different subtypes of P2Y receptor expressed in cells of the CNS that are activated by a select group of nucleotide agonists. Here, the agonist selectivity of these 8 P2Y receptor subtypes is reviewed with an emphasis on synthetic agonists with high potency and resistance to degradation by extracellular nucleotidases that have potential applications as therapeutic agents. In addition, the recent identification of a wide variety of subtype-selective antagonists is discussed, since these compounds are critical for discerning cellular responses mediated by activation of individual P2Y receptor subtypes. The functional expression of P2Y receptor subtypes in cells that comprise the CNS is also reviewed and the role of each subtype in the regulation of physiological and pathophysiological responses is considered. Other topics include the role of P2Y receptors in the regulation of blood-brain barrier integrity and potential interactions between different P2Y receptor subtypes that likely impact tissue responses to extracellular nucleotides in the CNS. Overall, current research suggests that P2Y receptors in the CNS regulate repair mechanisms that are triggered by tissue damage, inflammation and disease and thus P2Y receptors represent promising targets for the treatment of neurodegenerative diseases.

Extracellular nucleotide and nucleoside are signaling molecules with a wide range of actions in the central nervous system (CNS). Extracellular ATP is released by several mechanisms involving ATP binding cassette transporters, hemichannels, P2X7 receptors, or volume-sensitive chloride channels. The levels of ATP and its hydrolysis product, adenosine, in the synaptic cleft are controlled by a complex cascade of cell surface-located enzymes collectively known as ectonucleotidases. There are four major families of ectonucleotidases: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs), alkaline phosphatases, and ecto-5'- nucleotidase. Besides the production of adenosine through nucleotide hydrolysis, this neuromodulator can be released as adenosine per se by equilibrative and/or concentrative nucleoside transporters. In this review, the involvement of nucleotide/nucleoside transporters and ectonucleotidases in the pathophysiology of brain disorders is discussed. The identification of compounds able to modulate the activity of these players in purinergic neurotransmission and their implications in neurological disorders as potential targets for drug discovery is also highlighted.

While ATP is recognized as an intracellular energy source for many biochemical reactions, it is now recognised it is also an important extracellular signalling molecule. ATP is involved in both physiological and pathological events in most cell types, and receptor subtypes have been cloned and characterised. An important goal of purinergic research today is to annotate the human genome with functional information regarding the role of genes for purinergic receptors, ectonucleotidases and transporters, in brain physiology and pathology. Insights into these roles have been gained also from studies of the various purinergic knockouts, and here we report on the generation of these purinergic receptor/ectonucleotidase-null mice. Recent X-ray structures of purinergic ligand-activated receptors provide promising templates to understand the molecular mechanism of receptor actions at the atomic level, and to deploy X-ray structures to be used for structure-based drug design. In the present work we also summarize recent findings about X-ray structures of ionotropic and metabotropic purinergic receptors and ectonucleotidases. A novel and prominent role as modulators of signal propagation in animal cells is played by microRNAs. By acting as genetic switches, they might become stringent regulators of the variety of cellular responses triggered by the dynamic interactions between purinergic receptors, nucleotides/nucleosides, transporters and ectonucleotidases. In this review we highlight data on the regulation of purinergic mechanisms by microRNAs. Finally, we would like to illustrate what information is still missing or needed for the acquisition of a more complete knowledge of purinergic signalling.

Nurr1 is a member of the nuclear receptor superfamily and is a potential susceptibility gene for Parkinson’s disease (PD). Several lines of studies in vitro and in vivo reported that defects in the Nurr1 gene cause nigrostriatal neuronal deficiency as seen in PD. In the present study, we used a a synthetic low molecular weight Nurr1 activator which increases the transcription of Nurr1 to investigate whether it has anti-parkinsonian effects against nigrostriatal neuronal degeneration induced by proteasome inhibitor lactacystin. Adult C57BL/6 mice were treated orally with the Nurr1 activator and an inactive structural analog as a control at a dose of 10mg/kg per day, starting 3 days before microinjection of proteasome inhibitor lactacystin into the medial forebrain bundle and the treatment continued for a total of 4 weeks. Animal behavior tests, and pathological and biochemical examinations were performed to determine the anti-parkinsonian effects of the Nurr1 activator. We found that treatment with the Nurr1 activator significantly improved rotarod performance, attenuated dopamine neuron loss and nigrostriatal dopamine reduction, increased expression of Nurr1, dopamine transporter and vesicular monoamine transporter 2, and alleviated microglial activation in the substantia nigra of lactacystin-lesioned mice. These results suggest that the Nurr1 activator may become an innovative strategy for the treatment of PD.

This study explores the ability of a catalytic antioxidant, Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), to protect against neuronal and glial oxidative stress and death after spinal cord injury (SCI). Nine different doses of MnTBAP were administered into the intrathecal space of the rat spinal cord immediately following moderate SCI to establish dose - response curves for prevention of lipid peroxidation and neuron death. An optimal dose was determined by comparing the effectiveness of MnTBAP protection among doses. The optimal dose was then administered and the cords were removed 24 h post-administration and processed for staining. The cells in the cord sections at different distances from the epicenter were counted to obtain the spatial profiles of MnTBAP protection. Comparison of the counts between MnTBAP- and vehicle-treated groups in the sections double immuno-fluorescence-stained with oxidative and cellular markers demonstrated that MnTBAP significantly reduced numbers of nitrotyrosine- and DNP-positive (stained with an antibody against 2,4-dinitrophenyl hydrazine (DNPH)-labeled protein carbonyls) neurons, astrocytes, and oligodendrocytes. Comparison of the counts between the two treatments in the sections immuno-stained with cellular markers revealed that MnTBAP significantly increased numbers of neurons, motoneurons, astrocytes, and oligodendrocytes. MnTBAP more effectively reduced neuronal than glial cell death. Post-injury treatment with the optimal dose of MnTBAP at 6, 12, 24, 48, and 72 h post-SCI demonstrated that the effective time window for reducing protein nitration and neuron death was at least 12 h. Our results demonstrated that MnTBAP combats oxidative stress, thereby attenuating all types of cell death after SCI.

Methylene Blue (MB) is being investigated in clinical studies for its beneficial effects in the treatment of Alzheimer disease. However, its exact mechanisms of action have not been fully elucidated. The modulation of nicotinic acetylcholine receptors (nAChRs) has been suggested to play a role in the pathogenesis of various neurodegenerative diseases. Therefore, in the present study, the effect of MB on the function of the cloned α7 subunit of the human nAChR expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp technique. MB reversibly inhibited ACh (100 μM)-induced currents in a concentration-dependent manner with an IC50 value of 3.4 ± 0.3 μM. The effect of MB was not dependent on the membrane potential. MB did not affect the activity of endogenous Ca2+-dependent Cl- channels, since the inhibition by MB was unaltered in oocytes injected with the Ca2+ chelator 1,2-bis (o-aminophenoxy) ethane-N, N, N’, N’-tetraacetic acid and perfused with Ca2+-free bathing solution containing 1.8 mM Ba2+. MB decreased the maximal ACh-induced responses without significantly affecting ACh potency. Furthermore, specific binding of [125I] α-bungarotoxin, a radioligand selective for the α7 nAChR, was not altered by MB (10 μM), indicating that MB acts as a noncompetitive antagonist on α7 nAChRs. In hippocampal slices, whole-cell recordings from CA1 pyramidal neurons indicated that the increases in the frequency and amplitudes of the γ-aminobutyric acid-mediated spontaneous postsynaptic currents induced by bath application of 2 mM choline, a specific agonist for α7 nAChRs, were abolished after 10 min application of 3 μM MB. These results demonstrate that MB inhibits the function of human α7 nAChRs expressed in Xenopus oocytes and of α7 nAChR-mediated responses in rat hippocampal neurons.