Nadia D'Ambrosi

Nucleotide-mediated calcium signaling in rat cortical astrocytes: Role of P2X and P2Y receptors

ATP is the dominant messenger for astrocyte‐to‐astrocyte calcium‐mediated communication. Definition of the exact ATP/P2 receptors in astrocytes and of their coupling to intracellular calcium ([Ca2+]i) has important implications for brain physiology and pathology. We show that, with the only exception of the P2X6 receptor, primary rat cortical astrocytes express all cloned ligand‐gated P2X (i.e., P2X1–5 and P2X7) and G‐protein‐coupled P2Y receptors (i.e., P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12). These cells also express the P2Y‐like UDP‐glucose receptor, which has been recently recognized as the P2Y14 receptor. Single‐cell image analysis showed that only some of these receptors are coupled to [Ca2+]i. While ATP induced rapid and transient [Ca2+]i increases (counteracted by the P2 antagonists suramin, pyridoxal‐phosphate‐6‐azophenyl‐2′‐4′‐disulfonic acid and oxidized ATP), the P2X1/P2X3 agonist αβmeATP produced no changes. Conversely, the P2X7 agonist BzATP markedly increased [Ca2+]i; the presence and function of the P2X7 receptor was also confirmed by the formation of the P2X7 pore. ADP and 2meSADP also produced [Ca2+]i increases antagonized by the P2Y1 antagonist MRS2179. Some cells also responded to UTP but not to UDP. Significant responses to sugar‐nucleotides were also detected, which represents the first functional response reported for the putative P2Y14 receptor in a native system. Based on agonist preference of known P2 receptors, we conclude that, in rat astrocytes, ATP‐induced calcium rises are at least mediated by P2X7 and P2Y1 receptors; additional receptors (i.e., P2X2, P2X4, P2X5, P2Y2, P2Y4, and P2Y14) may also contribute.

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.

Extracellular ATP and neurodegeneration.

ATP is a potent signaling molecule abundantly present in the CNS. It elicits a wide array of physiological effects and is regarded as the phylogenetically most ancient epigenetic factor playing crucial biological roles in several different tissues. These can range from neurotransmission, smooth muscle contraction, chemosensory signaling, secretion and vasodilatation, to more complex phenomena such as immune responses, pain, male reproduction, fertilization and embryonic development. ATP is released into the extracellular space either exocytotically or from damaged and dying cells. It is often co-released with other neurotransmitters and it can interact with growth factors at both receptor- and/or signal transduction-level. Once in the extracellular environment, ATP binds to specific receptors termed P2. Based on pharmacological profiles, on selectivity of coupling to second-messenger pathways and on molecular cloning, two main subclasses with multiple subtypes have been distinguished. They are P2X, i.e. fast cation-selective receptor channels (Na+, K+, Ca2+), possessing low affinity for ATP and responsible for fast excitatory neurotransmission, and P2Y, i.e. slow G protein-coupled metabotropic receptors, possessing higher affinity for the ligand. In the nervous system, they are broadly expressed in both neurons and glial cells and can mediate dual effects: short-term such as neurotransmission, and long-term such as trophic actions. Since massive extracellular release of ATP often occurs after metabolic stress, brain ischemia and trauma, purinergic mechanisms are also correlated to and involved in the etiopathology of many neurodegenerative conditions. Furthermore, extracellular ATP per se is toxic for primary neuronal dissociated and organotypic CNS cultures from cortex, striatum and cerebellum and P2 receptors can mediate and aggravate hypoxic signaling in many CNS neurons. Conversely, several P2 receptor antagonists abolish the cell death fate of primary neuronal cultures exposed to excessive glutamate, serum/potassium deprivation, hypoglycemia and chemical hypoxia. In parallel with these detrimental effects, also trophic functions have been extensively described for extracellular purines (both for neuronal and non-neuronal cells), but these might either aggravate or ameliorate the normal cellular conditions. In summary, extracellular ATP plays a very complex role not only in the repair, remodeling and survival occurring in the nervous system, but even in cell death and this can occur either after normal developmental conditions, after injury, or acute and chronic diseases.

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.

The Proinflammatory Action of Microglial P2 Receptors Is Enhanced in SOD1 Models for Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of lower and upper motoneurons. The pathology is imputable in ∼2% of cases to mutations in the ubiquitous enzyme Cu, Zn superoxide dismutase (SOD1). Common theories to explain the pathogenic mechanisms of ALS include activation of microglia, responsible for the release of proinflammatory factors. However, how mutant SOD1 affects microglial activation and subsequently injures neurons is still unclear. Considering that extracellular ATP, through purinergic P2 receptors, constitutes a well recognized neuron-to-microglia alarm signal, the aim of this study was to investigate how the expression of mutant SOD1 affects P2 receptor-mediated proinflammatory microglial properties. We used primary and immortalized microglial cells from mutant SOD1 mice to explore several aspects of activation by purinergic ligands and to analyze the overall effect of such stimulation on the viability of NSC-34 and SH-SY5Y neuronal cell lines. We observed up-regulation of P2X4, P2X7, and P2Y6 receptors and down-regulation of ATP-hydrolyzing activities in mutant SOD1 microglia. This potentiation of the purinergic machinery reflected into enhanced sensitivity mainly to 2′-3′-O-(benzoyl-benzoyl) ATP, a P2X7 receptor preferential agonist, and translated into deeper morphological changes, enhancement of TNF-α and cyclooxygenase-2 content, and finally into toxic effects exerted on neuronal cell lines by microglia expressing mutant SOD1. All these parameters were prevented by the antagonist Brilliant Blue G. The purinergic activation of microglia may thus constitute a new route involved in the progression of ALS to be exploited to potentially halt the disease.

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.

The NADPH Oxidase Pathway Is Dysregulated by the P2X7 Receptor in the SOD1-G93A Microglia Model of Amyotrophic Lateral Sclerosis

Inflammation and oxidative stress are thought to play determinant roles in the pathogenesis of amyotrophic lateral sclerosis (ALS). Degenerating motor neurons produce signals that activate microglia to release reactive oxygen species (ROS) and proinflammatory cytokines, resulting in a vicious cycle of neurodegeneration. The ALS-causing mutant protein Cu+/Zn+ superoxide dismutase SOD1-G93A directly enhances the activity of the main ROS-producing enzyme in microglia, NADPH oxidase 2 (NOX2), a well-known player in the pathogenesis of ALS. Considering that extracellular ATP through P2X7 receptor constitutes a neuron-to-microglia alarm signal implicated in ALS pathology, we used primary microglial cells derived from transgenic SOD1-G93A mice and SOD1-G93A mice lacking the P2X7 receptor to investigate the effects of both pharmacological induction and genetic ablation of receptor activity on the NOX2 pathway. We observed that, in SOD1-G93A microglia, the stimulation of P2X7 receptor by 2′-3′-O-(benzoyl-benzoyl) ATP enhanced NOX2 activity in terms of translocation of p67phox to the membrane and ROS production; this effect was totally dependent on Rac1. We also found that, following P2X7 receptor stimulation, the phosphorylation of ERK1/2 was augmented in ALS microglia, and there was a mutual dependency between the NOX2 and ERK1/2 pathways. All of these microglia-mediated damaging mechanisms were prevented by knocking out P2X7 receptor and by the use of specific antagonists. These findings suggest a noxious mechanism by which P2X7 receptor leads to enhanced oxidative stress in ALS microglia and identify the P2X7 receptor as a promising target for the development of therapeutic strategies to slow down the progression of ALS.

Ablation of P2X7 receptor exacerbates gliosis and motoneuron death in the SOD1-G93A mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurological disorder characterized by selective degeneration of upper and lower motoneurons. The primary triggers for motoneuron degeneration are still unknown, but inflammation is considered an important contributing factor. P2X7 receptor is a key player in microglia response to toxic insults and was previously shown to increase pro-inflammatory actions of SOD1-G93A ALS microglia. We therefore hypothesized that lack of P2X7 receptor could modify disease features in the SOD1-G93A mice. Hetero- and homozygous P2X7 receptor knock-out SOD1-G93A mice were thus generated and analysed for body weight, disease onset and progression (by behavioural scores, grip and rotarod tests) and survival. Although the lifespan of P2X7(+/-) and P2X7(-/-)/SOD1-G93A female mice was extended by 6-7% with respect to SOD1-G93A mice, to our surprise the clinical onset was significantly anticipated and the disease progression worsened in both male and female P2X7(-/-)/SOD1-G93A mice. Consistently, we found increased astrogliosis, microgliosis, motoneuron loss, induction of the pro-inflammatory markers NOX2 and iNOS and activation of the MAPKs pathway in the lumbar spinal cord of end-stage P2X7(-/-)/SOD1-G93A mice. These results show that the constitutive deletion of P2X7 receptor aggravates the ALS pathogenesis, suggesting that the receptor might have beneficial effects in at least definite stages of the disease. This study unravels a complex dual role of P2X7 receptor in ALS and strengthens the importance of a successful time window of therapeutic intervention in contrasting the pathology.

Overexpression of superoxide dismutase 1 protects against β-amyloid peptide toxicity: effect of estrogen and copper chelators

Beta-amyloid peptides (Abeta) are major constituents of senile plaques in Alzheimers disease (AD) brain and contribute to neurodegeneration, operating through activation of apoptotic pathways. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. Estrogen is thought to play a protective role against neurodegeneration through a variety of mechanisms including scavenging of reactive oxygen species (ROS). In this study, we have challenged with Abeta, either in the presence or in the absence of 17beta-estradiol, differentiated human neuroblastoma SH-SY5Y cells (named line SH) and the same line overexpressing anti-oxidant enzyme superoxide dismutase 1 (SOD1; named line WT). We have observed that: (1) WT cells are less susceptible than SH cells to Abeta insult; (2) caspase-3, but not caspase-1, is involved in Abeta-induced apoptosis in this system; (3) estrogen protects both lines, without significantly affecting SOD activity; and (4) copper chelators prevent Abeta-induced toxicity. Our results further support the notion that anti-oxidant therapy might be beneficial in the treatment of AD by preventing activation of selected apoptotic pathways.