Cinzia Volonté

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.

P2X7 receptor modulation on microglial cells and reduction of brain infarct caused by middle cerebral artery occlusion in rat.

Adenosine 5′-triphosphate outflow increases after an ischemic insult in the brain and may induce the expression of P2X7 receptors in resting microglia, determining its modification into an activated state. To assess the effects of P2X7 receptor blockade in preventing microglia activation and ameliorating brain damage and neurological impairment, we delivered the P2 unselective antagonist Reactive Blue 2 to rats after middle cerebral artery occlusion. In sham-operated animals, devoid of brain damage, double immunofluorescence verified the absence of P2X7 immunoreactivity on resting microglia, astrocytes, and neurons, identified, respectively, by OX-42, glial fibrillary acid protein, and neuronal nuclei (NeuN) immunoreactivity. After ischemia, vehicle-treated rats showed monolateral sensorimotor deficit and tissue damage in striatum and frontoparietal cortex. Moreover, P2X7 immunoreactivity was de novo expressed on activated microglia in infarcted and surrounding areas, as well as on a reactive form of microglia, resting in shape but P2X7 immunoreactive, present in ipsi- and contralateral cingulate and medial frontal cortex. Reactive Blue 2 improved sensorimotor deficit and restricted the volume of infarction, without preventing the expression of P2X7, but inducing it in the microglia of contralateral frontal and parietal cortex and striatum, which had lost reciprocal connections with the remote infarct area. De novo expression of P2X7 occurred in both activated and reactive microglia, suggesting their differentiated roles in the area of infarct and in remote regions. Reactive Blue 2 reduced ischemic brain damage, likely blocking the function of activated microglia in the infarct area, but in the remote brain regions promoted the expression of P2X7 on reactive microglia, developing defense and reparative processes.

Membrane compartments and purinergic signalling: the purinome, a complex interplay among ligands, degrading enzymes, receptors and transporters

Receptors should be properly analysed in view of the microenvironment in which they are embedded. Therefore, the concept of ‘receptosome’ was formulated to the complex interactions taking place between receptors and other proteins at the plasma membrane level, and to explain very heterogeneous or divergent cellular responses to common epigenetic factors and modifications to the extracellular environment. The receptosome thus becomes a molecular network connecting transmitters, hormones or growth factors, to both their specific receptors and unique downstream effector proteins. As an example of receptosome, we introduce here the ‘purinome’ as molecular complex responsible for the biological effects of extracellular purine and pyrimidine ligands. In addition to a vast heterogeneity of purinergic ligands, the purinome thus consists of ectonucleotide‐metabolizing enzymes hydrolysing nucleoside phosphates, purinergic receptors classified as P1 for adenosine/AMP and P2 for nucleosides tri‐/diphosphates, nucleoside transporters with both equilibrative and concentrative properties and finally, nucleotide channels and transporters. Notably, these purinergic elements are not independent, but they play tightly concerted actions under physiological conditions. As a whole and not singularly, they trigger, maintain and terminate the purinergic signalling. This signifies that the purinome is not a new, mere definition of juxtaposed purinergic units, but rather the experimental evidence of complex and dynamic molecular cross‐talk and cooperation networks. Alteration of this dynamic equilibrium may even participate in many pathological states. As a consequence, to be successful against pathological conditions, the genetic/pharmacological manipulation of purinergic mechanisms must go well beyond single proteins, and be more holistically oriented.

Synaptic P2X7 and Oxygen/Glucose Deprivation in Organotypic Hippocampal Cultures

The P2X7 receptor for extracellular ATP is the main candidate, among P2 receptors, inducing cell death in the immune system. Here, we demonstrate the direct participation of this receptor to cell damage induced by oxygen/glucose deprivation, in the ex vivo model of organotypic hippocampal cultures. By pharmacological and immunological approaches, we show that P2X7 is rapidly and transiently up regulated in hippocampal areas eliciting metabolism impairment. Moreover, the P2 antagonists 2′,3′,-dialdehyde ATP and reactive blue 2 prevent both up regulation of this receptor and hypoxic/hypoglycemic damage. By confocal laser microscopy, we show that P2X7 is present at the synaptic level of fibers extending from the CA1–2 pyramidal cell layer throughout the strata oriens and radiatum, but absent on oligodendrocytes, astrocytes or neuronal cell bodies. Colocalization of P2X7 is obtained with neurofilament-L protein and with synaptophysin, not with myelin basic protein, glial fibrillary acidic protein or a marker for neuronal nuclei. P2X7 up regulation and diffuse cellular damage are also induced by 3′-O-(4-benzoyl) benzoyl-ATP, an agonist selective but not exclusive for P2X7. In summary, our study demonstrates that P2X7 not only directly participates to the hypoxic/hypoglycemic process, but also owns specific phenotypic localization. We do not exclude that it might serve as a sensor of dysregulated neuronal activity and ATP release, both occurring during oxygen/glucose deprivation.

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.

P2X3 receptor localizes into lipid rafts in neuronal cells.

P2X receptors are a family of seven (P2X1–7) cation channels gated by extracellular ATP, widely expressed in neurons and nonneuronal cells. Lipid rafts are cholesterol/sphingolipid‐rich membrane domains, involved in many cellular processes, including transmembrane receptor signaling, vesicle traffic, and protein sorting. We provide direct biochemical evidence that P2X3 receptor localizes into lipid rafts, in primary cultures of cerebellar granule neurons as well as in brain and dorsal root ganglia extracts. We show that P2X3 exhibits all the characteristics distinctive of a protein associated with lipid rafts. These characteristics include resistance to detergent extraction at 4°C, solubility after extraction of cholesterol from membranes with either saponin or methyl‐β‐cyclodextrin, and partitioning to low buoyant density fractions after sucrose gradient centrifugation in both detergent‐containing and detergent‐free conditions. Furthermore, P2X3 localizes in raft‐containing fractions in transiently transfected SH‐SY5Y neuroblastoma cells. The present finding contributes to the characterization of the functional localization of P2X3 in neurons and provides a novel potential mechanism for correct targeting and dynamic activation of this receptor.

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.

P2Y12 Receptor Protein in Cortical Gray Matter Lesions in Multiple Sclerosis

Although Multiple Sclerosis (MS) is regarded as a white matter disease, the incidence of demyelination and axonal injury is prominent also in gray matter. In MS, extracellular adenosine triphosphate (ATP) is an important mediator of central nervous system pathology via its ability to cause oligodendrocyte excitotoxicity. We have analyzed the distribution pattern of all ionotropic P2X and metabotropic P2Y receptors for ATP in postmortem samples of the cerebral cortex from healthy human subjects as well as MS patients. We focus particularly on the P2Y(12) subtype that is highly enriched in oligodendrocytes. We correlate the expression of this receptor to the extent of gray matter demyelination and pathological alterations occurring during secondary progressive MS. Using triple immunofluorescence and confocal analysis, we show that in sections of cerebral cortex from postmortem MS brains, the P2Y(12) protein is present in myelin and interlaminar astrocytes but absent from protoplasmic astrocytes residing in the deeper cortical layers, from microglia/macrophages, and from intact demyelinated axons. We report that a decreased P2Y(12) receptor immunoreactivity in proximity to the lesions is directly correlated with the extent of demyelination found in all types of gray matter cortical plaques (I-III) and subcortical white matter. Our study provides further insights into the pathogenetic features of MS and suggests that the loss of purinergic P2Y(12) receptors might be detrimental to tissue integrity.

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.