MicroRNA-125b regulates microglia activation and motor neuron death in ALS.

Understanding the means by which microglia self-regulate the neuroinflammatory response helps modulating their reaction during neurodegeneration. In amyotrophic lateral sclerosis (ALS), classical NF-κB pathway is related to persistent microglia activation and motor neuron injury; however, mechanisms of negative control of NF-κB activity remain unexplored. One of the major players in the termination of classical NF-κB pathway is the ubiquitin-editing enzyme A20, which has recognized anti-inflammatory functions. Lately, microRNAs are emerging as potent fine-tuners of neuroinflammation and reported to be regulated in ALS, for instance, by purinergic P2X7 receptor activation. In this work, we uncover an interplay between miR-125b and A20 protein in the modulation of classical NF-κB signaling in microglia. In particular, we establish the existence of a pathological circuit in which termination of A20 function by miR-125b strengthens and prolongs the noxious P2X7 receptor-dependent activation of NF-κB in microglia, with deleterious consequences on motor neurons. We prove that, by restoring A20 levels, miR-125b inhibition then sustains motor neuron survival. These results introduce miR-125b as a key mediator of microglia dynamics in ALS.

The complex folding behavior of HIV-1-protease monomer revealed by optical-tweezer single-molecule experiments and molecular dynamics simulations.

We have used optical tweezers and molecular dynamics simulations to investigate the unfolding and refolding process of a stable monomeric form of HIV-1-protease (PR). We have characterized the behavior under tension of the native state (N), and that of the ensemble of partially folded (PF) conformations the protein visits en route to N, which collectively act as a long-lived state controlling the slow kinetic phase of the folding process. Our results reveal a rich network of unfolding events, where the native state unfolds either in a two-state manner or by populating an intermediate state I, while the PF state unravels through a multitude of pathways, underscoring its structural heterogeneity. Refolding of mechanically denatured HIV-1-PR monomers is also a multiple-pathway process. Molecular dynamics simulations allowed us to gain insight into possible conformations the protein adopts along the unfolding pathways, and provide information regarding possible structural features of the PF state.

Dysregulated microRNAs in amyotrophic lateral sclerosis microglia modulate genes linked to neuroinflammation.

MicroRNAs (miRNAs) regulate gene expression at post-transcriptional level and are key modulators of immune system, whose dysfunction contributes to the progression of neuroinflammatory diseaseas such as amyotrophic lateral sclerosis (ALS), the most widespread motor neuron disorder. ALS is a non-cell-autonomous disease targeting motor neurons and neighboring glia, with microgliosis directly contributing to neurodegeneration. As limited information exists on miRNAs dysregulations in ALS, we examined this topic in primary microglia from superoxide dismutase 1-G93A mouse model. We compared miRNAs transcriptional profiling of non-transgenic and ALS microglia in resting conditions and after inflammatory activation by P2X7 receptor agonist. We identified upregulation of selected immune-enriched miRNAs, recognizing miR-22, miR-155, miR-125b and miR-146b among the most highly modulated. We proved that miR-365 and miR-125b interfere, respectively, with the interleukin-6 and STAT3 pathway determining increased tumor necrosis factor alpha (TNFα) transcription. As TNFα directly upregulated miR-125b, and inhibitors of miR-365/miR-125b reduced TNFα transcription, we recognized the induction of miR-365 and miR-125b as a vicious gateway culminating in abnormal TNFα release. These results strengthen the impact of miRNAs in modulating inflammatory genes linked to ALS and identify specific miRNAs as pathogenetic mechanisms in the disease.

Metabotropic purinergic receptors in lipid membrane microdomains.

There is broad evidence that association of transmembrane receptors and signalling molecules with lipid rafts/caveolae provides an enriched environment for protein-protein interactions necessary for signal transduction, and a mechanism for the modulation of neurotransmitter and/or growth factor receptor function. Several receptors translocate into submembrane compartments after ligand binding, while others move in the opposite direction. The role of such a dynamic localization and functional facilitation is signalling modulation and receptor desensitization or internalization. Purine and pyrimidine nucleotides have been viewed as primordial precursors in the evolution of all forms of intercellular communication, and they are now regarded as fundamental extracellular signalling molecules. They propagate the purinergic signalling by binding to ionotropic and metabotropic receptors expressed on the plasma membrane of almost all cell types, tissues and organs. Here, we have illustrated the localization in lipid rafts/caveolae of G protein-coupled P1 receptors for adenosine and P2Y receptors for nucleoside tri- and di-phosphates. We have highlighted that microdomain partitioning of these purinergic GPCRs is cell-specific, as is the overall expression levels of these same receptors. Moreover, we have described that disruption of submembrane compartments can shift the purinergic receptors from raft/caveolar to non-raft/non-caveolar fractions, and then abolish their ability to activate lipid signalling pathways and to integrate with additional lipid-controlled signalling events. This modulates the biological response to purinergic ligands and most of all indicates that the topology of the various purinergic components at the cell surface not only organizes the signal transduction machinery, but also controls the final cellular response.

Purinergic signalling: what is missing and needed next? The use of transgenic mice, crystallographic analysis and MicroRNA.

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.

P2X7 receptors: channels, pores and more.

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-1ß, 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.

Do ATP and NO interact in the CNS?

Enzymatically derived NO and extracellular ATP are receiving greater attention due to their role as messengers in the CNS during different physiological and pathological processes. Ionotropic (P2XR) and metabotropic (P2YR) purinergic receptors mediate ATP effects and are present throughout the body. Particularly P2XR are crucial for brain plasticity mechanisms, and are involved in the pathogenesis of different CNS illnesses. NO does not have a specific receptor and its actions are directly dependent on the production on demand by different nitric oxide synthase isoforms. NO synthesizing enzymes are present virtually in all tissues, and NO influences multifarious physiological and pathological functions. Interestingly, various are the tissue and organs modulated by both ATP and NO, such as the immune, brain and vascular systems. Moreover, direct interactions between purinergic and nitrergic mechanisms outside the CNS are well documented, with several studies also indicating that ATP and NO do participate to the same CNS functions. In the past few years, further experimental evidence supported the physiological and pathological relevance of ATP and NO direct interactions in the CNS. The aim of the present review is to provide an account of the available information on the interplay between purinergic and nitrergic systems, focussing on the CNS. The already established relevance of ATP and NO in different pathological processes would predict that the knowledge of ATP/NO cross-talk mechanisms would support pharmacological approaches toward the development of novel ATP/NO combined pharmacological agents.

Oligodendrocytes express P2Y12 metabotropic receptor in adult rat brain.

In the CNS, nucleotide receptors termed P2 receptors are identified on neurons and glial cells, mediating neuron-neuron, glia-glia and glia-neuron communication. In the present work, we qualify in vivo in the adult rat CNS the cellular/subcellular distribution of P2Y12 receptor protein in cerebral cortex, white matter and subcortical nuclei (striatum and substantia nigra), by means of immunofluorescence-confocal, electron microscopy and Western blot analysis. P2Y12 receptor immunoreactivity colocalizes neither with markers such as neuronal nuclei, neurofilament light chain, calbindin and tyrosine hydroxylase, nor with glial fibrillary acidic protein and isolectin B4, but with myelin basic protein and the oligodendrocyte marker RIP, in both cell bodies and processes, indicating therefore oligodendrocyte localization. Electron microscopy identifies P2Y12 receptors in both the perikaryon and under the plasmalemma of oligodendrocyte cell bodies and radiating processes, until the paranodal region of fibers. By Western blot analysis, P2Y12 receptor shows a specific band of 42-44 kDa, matching the molecular mass predicted from amino acid sequencing. Since in platelets P2Y12 receptor is known to regulate adhesion/activation and thrombus growth/stability, from our results we could speculate by analogy that, in oligodendrocytes, P2Y12 receptor signaling might contribute to the migration and adhesion of the glial processes to axons to be myelinated.

ATP regulates oligodendrocyte progenitor migration, proliferation, and differentiation: involvement of metabotropic P2 receptors.

Extracellular nucleotides act as potent signaling molecules in the neuron-glia and glia-glia communication, via the activation of specific ligand-gated P2X and G-protein-coupled metabotropic P2Y receptors. Most of the data available about the effects of P2 receptor activation in the CNS concern astrocytes, microglia, and neurons. To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in rat oligodendrocyte progenitors (OPs) and investigated the effects of ATP and its breakdown products on their functions. We describe here that rat OPs express different types of P2 receptors and that nucleotide-induced Ca(2+) raises in these progenitor cells are mainly due to the activation of P2X(7) ionotropic and ADP-sensitive P2Y(1) metabotropic receptors. We also show that ATP and ADP stimulate OP migration, inhibit the mitogenic response of OPs to PDGF and promote oligodendrocyte differentiation. The pharmacological profile of the nucleotide-induced effects demonstrates the important regulatory role of P2Y(1) receptor signaling in OP functions. These findings suggest that ATP, which is released in high amounts under inflammatory conditions and following cell death, might regulate remyelination processes in inflammatory demyelinating diseases of the CNS, like multiple sclerosis.

Metabotropic P2 receptor activation regulates oligodendrocyte progenitor migration and development.

To gain insights into the role of purinergic receptors in oligodendrocyte development, we characterized the expression and functional activity of P2 receptors in cultured rat oligodendrocyte progenitors and investigated the effects of ATP and its breakdown products on the migration and proliferation of this immature glial cell population. Using Western blot analysis, we show that oligodendrocyte progenitors express several P2X (P2X(1,2,3,4,7)) and P2Y (P2Y(1,2,4)) receptors. Intracellular Ca(2+) recording by Fura-2 video imaging allowed to determine the rank potency order of the P2 agonists tested: ADPbetaS = ADP = Benzoyl ATP > ATP > ATPgammaS > UTP, alpha,beta-meATP ineffective. Based on the above findings, on pharmacological inhibition by the antagonists oxATP and MRS2179, and on the absence of alpha,betameATP-induced inward current in whole-cell recording, P2X(7) and P2Y(1) were identified as the main ionotropic and metabotropic P2 receptors active in OPs. As a functional correlate of these findings, we show that ATP and, among metabotropic agonists, ADP and the P2Y(1)-specific agonist ADPbetaS, but not UTP, induce oligodendrocyte progenitor migration. Moreover, ATP and ADP inhibited the proliferation of oligodendrocyte progenitors induced by platelet-derived growth factor, both in purified cultures and in cerebellar tissue slices. The effects of ATP and ADP on cell migration and proliferation were prevented by the P2Y(1) antagonist MRS2179. By confocal laser scanning microscopy, P2Y(1) receptors were localized in NG2-labeled oligodendrocyte progenitors in the developing rat brain. These data indicate that ATP and ADP may regulate oligodendrocyte progenitor functions by a mechanism that involves mainly activation of P2Y(1) receptors.

Up-regulation of P2X2, P2X4 receptor and ischemic cell death: prevention by P2 antagonists.

In the present work we examined the involvement of selected P2X receptors for extracellular ATP in the onset of neuronal cell death caused by glucose/oxygen deprivation. The in vitro studies of organotypic cultures from hippocampus evidenced that P2X2 and P2X4 were up-regulated by glucose/oxygen deprivation. Moreover, we showed that ischemic conditions induced specific neuronal loss not only in hippocampal, but also in cortical and striatal organotypic cultures and the P2 receptor antagonists basilen blue and suramin prevented these detrimental effects. In the in vivo experiments we confirmed the induction of P2X receptors in the hippocampus of gerbils subjected to bilateral common carotid occlusion. In particular, P2X2 and P2X4 proteins became significantly up-regulated, although to different extent and in different cellular phenotypes. The induction was confined to the pyramidal cell layer of the CA1 subfield and to the transition zone of the CA2 subfield and it was coincident with the area of neuronal damage. P2X2 was expressed in neuronal cell bodies and fibers in the CA1 pyramidal cell layer and in the strata oriens and radiatum. Intense P2X4 immunofluorescence was localized to microglia cells. Our results indicate a direct involvement of P2X receptors in the mechanisms sustaining cell death evoked by metabolism impairment and suggest the use of selected P2 antagonists as effective neuroprotecting agents.

Cerebellar lesion up-regulates P2X1 and P2X2 purinergic receptors in precerebellar nuclei.

ATP released in the extracellular space by neuronal injury can influence neighboring neurons via activation of purinergic receptors. In vitro data suggest the involvement of ATP and purinergic receptors as trophic agents in different biological events such as neuritogenesis and cell survival. Recently, in vivo studies have demonstrated modifications in the glial expression of ionotropic purinergic receptors after CNS lesions. In the present study, we investigated the effects of CNS lesion on the neuronal expression of P2X(1) and P2X(2) receptor subunits by immunohistochemistry and western blotting techniques. In the precerebellar structures of normal animals the expression of P2X(1) and P2X(2) was lower than previously reported. P2X(1) immunostaining was confined only to fibers, while P2X(2) immunostaining demonstrated a neuronal expression. After unilateral cerebellar lesion (hemicerebellectomy) axotomized precerebellar neurons underwent marked cell loss; however, some precerebellar neurons did not degenerate. Seven to 35 days after hemicerebellectomy, a transient, time-dependent, marked increase in the number of immunopositive P2X(1) and P2X(2) neurons was observed in the precerebellar nuclei of the experimental side. An even distribution of immunopositive neurons was present in almost all precerebellar nuclei examined, except for the inferior olive. In this latter structure, differences in the distribution of immunopositive neurons were evident among the subnuclei. Up-regulation of immunoreactivity over relatively long time periods, distribution selectivity and absence of degenerating morphological features in immunopositive neurons suggest that purinergic receptors may have a role in mediating the survival of neuronal responses to axotomy. The present findings are the first report in the CNS of P2X(1) and P2X(2) receptor subunit involvement in neuronal reaction to axotomy. They provide in vivo evidence of a correlation between purinergic receptor subunit up-regulation and survival of injured neurons.

P2 receptor modulation and cytotoxic function in cultured CNS neurons.

In this study we investigate the presence, modulation and biological function of P2 receptors and extracellular ATP in cultured cerebellar granule neurons. As we demonstrate by RT-PCR and western blotting, both P2X and P2Y receptor subtypes are expressed and furthermore regulated as a function of neuronal maturation. In early primary cultures, mRNA for most of the P2 receptor subtypes, except P2X(6), are found, while in older cultures only P2X(3), P2Y(1) and P2Y(6) mRNA persist. In contrast, P2 receptor proteins are more prominent in mature neurons, with the exception of P2Y(1). We also report that extracellular ATP acts as a cell death mediator for fully differentiated and mature granule neurons, for dissociated striatal primary cells and hippocampal organotypic cultures, inducing both apoptotic and necrotic features of degeneration. ATP causes cell death with EC(50) in the 20-50 microM range within few minutes of exposure and with a time lapse of at most two hours. Additional agonists for P2 receptors induce toxic effects, whereas selected antagonists are protective. Cellular swelling, lactic dehydrogenase release and nuclei fragmentation are among the features of ATP-evoked cell death, which also include direct P2 receptor modulation. Comparably to P2 receptor antagonists previously shown preventing glutamate-toxicity, here we report that competitive and non-competitive NMDA receptor antagonists inhibit the detrimental consequences of extracellular ATP. Due to the massive extracellular release of purine nucleotides and nucleosides often occurring during a toxic insult, our data indicate that extracellular ATP can now be included among the potential causes of CNS neurodegenerative events.

Interaction between ATP and nerve growth factor signalling in the survival and neuritic outgrowth from PC12 cells.

In a previous study we used P2 receptor antagonists to inhibit diverse responses that nerve growth factor (NGF) promotes and coordinates in PC12 cells and we suggested that P2 receptors partake in the NGF signalling cascade. In this paper, we examine the direct role of extracellular P2 receptor agonists as neurotrophic factors. ATP and 2-Cl-ATP promote neurite regeneration after priming PC12 cells with NGF and the effect is dose-dependent, with an EC(50) of about 5 and 3 microM, respectively. The number of cell clumps bearing neurites was maximally induced in day 1 and it was maintained up to about one week by ATP, or up to at least 2 weeks by 2-Cl-ATP. The involvement of P1 receptors or intracellular inosine in these actions was excluded, whereas various antagonists of P2 receptors were inhibitory. Moreover, NGF and ATP caused a direct up-regulation of P2X(2), P2X(3), P2X(4) and P2Y(2), but not P2Y(4) receptor proteins under neurite-regenerating conditions, as well as extracellular signal-regulated kinase (Erk)1-2 tyrosine/threonine phosphorylation and activation. Finally, ATP, 2-Cl-ATP and ATPgammaS enhanced neurite initiation evoked by sub-optimal NGF concentrations and ATP and 2-Cl-ATP fully sustained survival of PC12 cells after serum deprivation. Our results establish that P2 receptor agonists can behave as neurotrophic factors for neuronal cells and suggest a potential interplay between ATP and NGF in the signalling pathways triggered on their target cells.

Glucose deprivation and chemical hypoxia: neuroprotection by P2 receptor antagonists.

In this work we investigate cell survival after glucose deprivation and/or chemical hypoxia and we analyse the neuroprotective properties of selected antagonists of P2 ATP receptors. We find that in rat cerebellar granule neurones, the antagonist basilen blue prevents neuronal death under hypoglycaemia. Basilen blue acts through a wide temporal range and it retains its efficacy under chemically induced hypoxic conditions, in the presence of the respiratory inhibitors of mitochondria electron transport chain complexes II (3-nitropropionic acid) and III (antimycin A). In spite of the presence of these compounds, basilen blue maintains normal intracellular ATP levels. It furthermore prevents neuronal death caused by agents blocking the mitochondrial calcium uptake (ruthenium red) or discharging the mitochondrial membrane potential (carbonyl cyanide m-chlorophenylhydrazone). Inhibition of poly (ADP-ribose) polymerase, modulation of the enzyme GAPDH and mitochondrial transport of mono-carboxylic acids are not conceivable targets for the action of basilen blue. Survival is sustained by basilen blue also in CNS primary cultures from hippocampus and in PNS sympathetic-like neurones. Partial neuroprotection is furthermore provided by three additional P2 receptor antagonists: suramin, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium and 4,4'-diisothiocyanatostilbene-2,2'disulphonic acid. Our data suggest the exploitation of selected P2 receptor antagonists as potential neuroprotective agents.

Hypoglycaemia-induced cell death: features of neuroprotection by the P2 receptor antagonist basilen blue.

Our previous work in neuronal cultures has shown that several antagonists of P2 ATP receptors prevent cell death evoked by hypoglycaemia, chemical hypoxia, mitochondria dysfunction, as well as glutamate-dependent excitotoxicity and low potassium-induced apoptosis. Experiments are now designed to examine which biological pathway contributes to cell death/survival under glucose starvation. We show here that, consequently to hypoglycaemic insults, cerebellar granule neurones undergo a combination of apoptosis and necrosis both inhibited by the P2 receptor antagonist basilen blue. This is demonstrated by morphological and biochemical features, such as TdT-mediated dUTP-biotin nick end-labelling, fluorescent staining of nuclear chromatin using Hoechst 33258, direct counting of intact viable nuclei and extracellular releasing of the cytosolic enzyme LDH. Furthermore, we show that hypoglycaemia induces outflow of cytochrome c from mitochondria and it up-regulates heat-shock proteins HSP70, but not HSP90, glucose-regulated proteins GRP75 and GRP78, as well as expression and activity of the enzyme caspase-2. Basilen blue can modulate only some of these effects. Our data contribute to dissect the role played by P2 receptor antagonism in sustaining neuroprotection against metabolic stresses.

Effect of P2 purinoceptor antagonists on kainate-induced currents in rat cultured neurons.

The action of purinergic antagonists on kainate-induced currents was studied in rat cortical neurons in primary culture using the whole-cell configuration of the patch-clamp technique. The amplitude of the currents induced by kainate in cortical neurons was concentration-dependent (EC(50)=106 microM). Pyridoxal-phosphate-6-azophenyll-2',4'-disulphonic acid 4-sodium (PPADS), a P2X antagonist, was ineffective in the reduction of the kainate-induced current in cortical neurons, while 2, 2'-pyridylisatogen (PIT), basilen blue (BB) and suramin, respectively two selective P2Y and a non-selective P2 receptor antagonist, caused a reduction in the amplitude of the current induced by kainate. BB decreased the inward current induced by kainate at all holding potentials and the reduction was dose-dependent (EC(50)=34 microM). The total conductance of the neurons for the kainate-induced current was significantly reduced (P<0.01) and the effect was completely reversible. BB furthermore reduced the kainate-induced current in granule and hippocampal neurons and decreased the amplitude of the alpha-amino-3-hydroxy-5-methyl-4-isoxalepropionic acid (AMPA)-evoked current in cortical neurons. Cholera toxin (ChTx) did not affect the action of BB on the kainate-induced currents in cortical neurons and moreover, when guanosine 5'-o-(3-thiotriphosphate) (GTPgammaS) was added to the electrode solution, the kainate-induced currents were still reduced by 100 microM BB. The maximal response to kainate decreased in the presence of 20 microM BB without changing its EC(50), indicating a non-competitive mechanism of inhibition. These results demonstrate that preferential P2Y receptor antagonists are able to modulate the kainate and AMPA-induced currents in central neurons, suggesting a potential use of these compounds as neuroprotective agents.

Antagonists of P2 receptor prevent NGF-dependent neuritogenesis in PC12 cells.

The pheochromocytoma PC12 cell line that develops neuronal characteristics of sympathetic cells after treatment with nerve growth factor (NGF) represents a well-established cellular model system for studying NGF signalling. Interesting information on the different mechanistic pathways of NGF can be obtained by adopting the pharmacological approach of inhibiting P2 receptors, expressed in naive PC12 cells and recognised as important biological mediators of neurotransmitters and growth factors. We show here that Basilen Blue, an antagonist of P2 receptor, reversibly prevents NGF-dependent neurite outgrowth with an IC(50) in the 5-10 microM range. Suramin, oxidised-ATP and diisothiocyanatostilbene-disulfonic acid, differently from other purinoceptor ligands, are also effective in this regard. NGF-dependent regeneration and stability of neurites, selected NGF-dependent extracellular and intracellular protein phosphorylations, binding of [(3)H] ATP to PC12 cell membranes are also modulated by Basilen Blue. On the contrary, cell adhesion, cellular duplication, 5'-nucleotidase activity, NGF-induced tyrosine autophosphorylation of TrkA receptors are not affected. NGF furthermore directly modulates the extracellular release of ATP and especially the levels of P2X(2) receptor protein in PC12 cells. In addition, extracellular ATP improves the neuritogenic effect of sub-optimal concentrations of NGF. Our study identifies P2 receptor ligands, particularly Basilen Blue, as useful tools to dissect different NGF-evoked functions, suggesting a mechanistic role for P2 receptors in the signalling pathways of NGF.

Neuroprotective effects of modulators of P2 receptors in primary culture of CNS neurones.

In previous studies (Volonté and Merlo, 1996. J. Neurosci. Res. 45, 183-193) basilen blue was shown to be a P2 receptor antagonist which abrogated glutamate-mediated cytotoxicity in cerebellar neurones in primary culture. Our work has now been extended to evaluate the neuroprotective action of the compound in additional neuronal systems, as well as in a different paradigm of cell death. We show that basilen blue prevents L-glutamate-mediated neurotoxicity in rat cerebellar (90-100% inhibition), cortical (60-70%) and hippocampal (50%) neurones. Similarly, glutamate-dependent progressive darkening of cell bodies, loss of phase-brightness and rapid cellular swelling are inhibited. Basilen blue is significantly less toxic and more effective at blocking L-glutamate toxicity in mixed cortical/glial cultures, compared to its structural analogue cibacron blue. Moreover, its neuroprotective effect is correlated with the time of incubation with granule neurones. Other purinoceptor ligands, including 2,2'-pyridylisatogen, but not pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium, are also effective in preventing glutamate toxicity. Furthermore, basilen blue prevents serum deprivation- and low potassium-induced apoptotic cell death in cerebellar granule neurones. In summary, our data extend and reinforce the possibility of a potential therapeutic use of P2 receptor modulators as neuroprotective agents for the central nervous system.