Chiara Cervetto

P2X7 pre-synaptic receptors in adult rat cerebrocortical nerve terminals: a role in ATP-induced glutamate release.

Although growing evidence suggests that extracellular ATP might play roles in the control of astrocyte/neuron crosstalk in the CNS by acting on P2X7 receptors, it is still unclear whether neuronal functions can be attributed to P2X7 receptors. In the present paper, we investigate the location, pharmacological profile, and function of P2X7 receptors on cerebrocortical nerve terminals freshly prepared from adult rats, by measuring glutamate release and calcium accumulation. The preparation chosen (purified synaptosomes) ensures negligible contamination of non‐neuronal cells and allows exposure of ‘nude’ release‐regulating pre‐synaptic receptors. To confirm the results obtained, we also carried out specific experiments on human embryonic kidney 293 cells which had been stably transfected with rat P2X7 receptors. Together, our findings suggest that (i) P2X7 receptors are present in a subpopulation of adult rat cerebrocortical nerve terminals; (ii) P2X7 receptors are localized on glutamatergic nerve terminals; (iii) P2X7 receptors play a significant role in ATP‐evoked glutamate efflux, which involves Ca2+‐dependent vesicular release; and (iv) the P2X7 receptor itself constitutes a significant Ca2+‐independent mode of exit for glutamate.

Carbenoxolone inhibits volume-regulated anion conductance in cultured rat cortical astroglia.

Accumulating evidence indicate that the gap-junction inhibitor carbenoxolone (CBX) regulates neuronal synchronization, depresses epileptiform activity and has a neuroprotective action. These CBX effects do not depend solely on its ability to inhibit gap junction channels formed by connexins (Cx), but the underlying mechanisms remain to be elucidated. Here we addressed the questions whether CBX modulates volume-regulated anion channels (VRAC) involved in the regulatory volume decrease and regulates the associated release of excitatory amino acids in cultured rat cortical astrocytes. We found that CBX inhibits VRAC conductance with potency comparable to that able to depress the activity of the most abundant astroglial gap junction protein connexin43 (Cx43). However, the knock down of Cx43 with small interfering RNA (siRNA) oligonucleotides and the use of various pharmacological tools revealed that VRAC inhibition was not mediated by interaction of CBX with astroglial Cx proteins. Comparative experiments in HEK293 cells stably expressing another putative target of CBX, the purinergic ionotropic receptor P2X7, indicate that the presence of this receptor was not necessary for CBX-mediated depression of VRAC. Finally, we show that in COS-7 cells, which are not endowed with pannexin-1 protein, another astroglial plasma membrane interactor of CBX, VRAC current retained its sensitivity to CBX. Complementary analyses indicate that the VRAC-mediated release of excitatory amino acid aspartate was decreased by CBX. Collectively, these findings support the notion that CBX could affect astroglial ability to modulate neuronal activity by suppressing excitatory amino acid release through VRAC. They also provide a possible mechanistic clue for the neuroprotective effect of CBX in vivo.

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X7 receptor (P2X7R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X7R activation induced Ca2+‐dependent vesicular glutamate release and significant Ca2+‐independent glutamate efflux through the P2X7R itself. In the present study, we investigated the effect of the new selective P2X7R competitive antagonist 3‐(5‐(2,3‐dichlorophenyl)‐1H‐tetrazol‐1‐yl)methyl pyridine (A‐438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca2+]i signals and glutamate release, and evaluated whether P2X7R immunoreactivity was consistent with these functional tests. A‐438079 inhibited functional responses. P2X7R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non‐glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X7R‐mediated [Ca2+]i signals. These findings provide compelling evidence of functional presynaptic P2X7R in cortical nerve terminals.

In vitro activation of GAT1 transporters expressed in spinal cord gliosomes stimulates glutamate release that is abnormally elevated in the SOD1/G93A(+) mouse model of amyotrophic lateral sclerosis

J. Neurochem. (2010) 113, 489–501.

Potentiation of NMDA Receptor-Dependent Cell Responses by Extracellular High Mobility Group Box 1 Protein

Background Extracellular high mobility group box 1 (HMGB1) protein can operate in a synergistic fashion with different signal molecules promoting an increase of cell Ca2+ influx. However, the mechanisms responsible for this effect of HMGB1 are still unknown. Principal Findings Here we demonstrate that, at concentrations of agonist per se ineffective, HMGB1 potentiates the activation of the ionotropic glutamate N-methyl-D-aspartate receptor (NMDAR) in isolated hippocampal nerve terminals and in a neuroblastoma cell line. This effect was abolished by the NMDA channel blocker MK-801. The HMGB1-facilitated NMDAR opening was followed by activation of the Ca2+-dependent enzymes calpain and nitric oxide synthase in neuroblastoma cells, resulting in an increased production of NO, a consequent enhanced cell motility, and onset of morphological differentiation. We have also identified NMDAR as the mediator of HMGB1-stimulated murine erythroleukemia cell differentiation, induced by hexamethylenebisacetamide. The potentiation of NMDAR activation involved a peptide of HMGB1 located in the B box at the amino acids 130–139. This HMGB1 fragment did not overlap with binding sites for other cell surface receptors of HMGB1, such as the advanced glycation end products or the Toll-like receptor 4. Moreover, in a competition assay, the HMGB1(130–139) peptide displaced the NMDAR/HMGB1 interaction, suggesting that it comprised the molecular and functional site of HMGB1 regulating the NMDA receptor complex. Conclusion We propose that the multifunctional cytokine-like molecule HMGB1 released by activated, stressed, and damaged or necrotic cells can facilitate NMDAR-mediated cell responses, both in the central nervous system and in peripheral tissues, independently of other known cell surface receptors for HMGB1.

Motor neuron dysfunction in a mouse model of ALS: gender-dependent effect of P2X7 antagonism.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative progressive currently untreatable disease, characterized by selective motor neuron degeneration; the incidence and prevalence of ALS are greater in men than in women. Although some important mechanisms that might contribute to the death of motor neurons have been identified, the mechanisms underlying disease pathophysiology are still uncertain. In particular, the mechanisms underlying the role of gender in ALS and whether treatments should take into account sexual dimorphism remain only partially understood. Recently, the P2X7 receptor for ATP was reported to display neurotoxic potential in motor neuron disorders, and antagonism of the receptor has been suggested to be helpful in these disorders. Studying transgenic mice with superoxide dismutase 1 gene mutations, widely used as model for ALS, may provide a better understanding of pathogenic mechanisms and of toxicity towards motor neurons, also possibly helping to understand whether treatments for ALS should take into account sexual dimorphism. The aim of the work was (1) investigating on gender-dependence of disease progression in the standard model for ALS - the transgenic mouse bearing superoxide dismutase 1 gene mutations - and (2) assessing if a P2X7 receptor antagonist treatment should take into account sexual dimorphism. We evaluated if gender affect the disease course, the motor performance, the weight loss and the lifespan in mice overexpressing mutant superoxide dismutase 1. We measured motor impairment, motor strength and coordination by rotarod and grip strength testing. Further, we assessed if a treatment with the P2X7 receptor antagonist Brilliant Blue G - a dye that can cross the blood-brain barrier, has low toxicity, and has exhibited therapeutic effects in animal models of neurodegenerative diseases - impact on the disease progression, in male and female ALS mice. We found that (1) the onset and the disease progression, and the survival were dependent on gender: male performed worst than female, lost body weight and died before; (2) treatment with the P2X7 receptor antagonist Brilliant Blue G ameliorated the disease progression. The treatment effect was gender-dependent: amelioration was greater in male than in female. In conclusions, we suggest that not only pathogenetic mechanism of motor neuron toxicity but also the drug treatment effectiveness may depend on gender; sexual dimorphism should be considered when investigating on ALS treatment efficacy in the ALS animal model. Our findings also point on the potential relevance of P2X7 receptor antagonism for ALS treatment, and highlight the importance of adopting a sex-specific approach to searching for treatment of ALS.

The P2X7 receptor as a route for non‐exocytotic glutamate release: dependence on the carboxyl tail

P2X7 receptors trigger Ca2+‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full‐length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18‐amino acid cysteine‐rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non‐competitive way by extracellular Mg2+, did not require the recruitment of pore‐forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine‐rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca2+ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca2+‐dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non‐exocytotic glutamate efflux.

Inhibition of Presynaptic Release-Facilitatory Kainate Autoreceptors by Extracellular Cyclic GMP

We found that both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate autoreceptors were present on the glutamate-releasing terminals of cerebellar parallel/climbing fibers and that they functioned as facilitatory autoreceptors. Extracellular cGMP inhibited the neurotransmitter release evoked by presynaptic kainate receptor activation; the inhibitory effect of extracellular cGMP was selective for the kainate autoreceptor-mediated response and did not affect the AMPA autoreceptor-mediated response. Endogenously synthesized cGMP might be the physiological source for the extracellular cGMP modulating the response to kainate autoreceptor activation.

Role of iso-receptors in receptor-receptor interactions with a focus on dopamine iso-receptor complexes

Abstract Intercellular and intracellular communication processes consist of signals and recognition/decoding apparatuses of these signals. In humans, the G protein-coupled receptor (GPCR) family represents the largest family of cell surface receptors. More than 30 years ago, it has been proposed that GPCR could form dimers or higher-order oligomers (receptor mosaics [RMs] at the plasma membrane level and receptor-receptor interactions [RRIs] have been proposed as a new integrative mechanism for chemical signals impinging on cell plasma membranes). The basic phenomena involved in RRIs are allostery and cooperativity of membrane receptors, and the present paper provides basic information concerning their relevance for the integrative functions of RMs. In this context, the possible role of iso-receptor RM is discussed (with a special focus on dopamine receptor subtypes and on some of the RMs they form with other dopamine iso-receptors), and it is proposed that two types of cooperativity, namely, homotropic and heterotropic cooperativity, could allow distinguishing two types of functionally different RMs. From a general point of view, the presence of iso-receptors and their topological organization within RMs allow the use of a reduced number of signals for the intercellular communication processes, since the target cells can recognize and decode the same signal in different ways. This theoretical aspect is further analyzed here by means of an analogy with artificial information systems. Thus, it is suggested that the ‘multiplexer’ and ‘demultiplexer’ concepts could, at least in part, model the role of RMs formed by iso-receptors in the information handling by the cell.

Oxaliplatin evokes P2X7-dependent glutamate release in the cerebral cortex: A pain mechanism mediated by Pannexin 1

Anticancer therapy based on the repeated administration of oxaliplatin is limited by the development of a neuropathic syndrome difficult to treat. Oxaliplatin neurotoxicity is based on complex nervous mechanisms, the comprehension of the role of single neurotransmitters and the knowledge of the signal flow among cells is matter of importance to improve therapeutic chances. In a rat model of oxaliplatin-induced neuropathy, we report increased P2X7-evoked glutamate release from cerebrocortical synaptosomes. The release was abolished by the P2X7 receptor (P2X7R) antagonists Brilliant-Blue-G (BBG) and A-438079, and significantly reduced by Carbenoxolone and the Pannexin 1 (Panx1) selective inhibitors Erioglaucine and (10)Panx suggesting the recruitment of Panx1. Aimed to evaluate the significance of P2X7R-Panx1 system activation in pain generated by oxaliplatin, pharmacological modulators were spinally infused by intrathecal catheter in oxaliplatin-treated animals. BBG, Erioglaucine and (10)Panx reverted oxaliplatin-dependent pain. Finally, the influence of the P2X7R-Panx1 system blockade on oxaliplatin anticancer activity was evaluated on the human colon cancer cell line HT-29. Prevention of HT-29 apoptosis and mortality was dependent by kind and concentration of P2X7R antagonists. On the contrary, the inhibition of Panx1 did not alter oxaliplatin lethality in tumor cells. It is concluded that glutamate release dependent on P2X7R is increased in cerebrocortical nerve terminals from oxaliplatin-treated rats; the increase is mediated by functional recruitment of Panx1; P2X7R antagonists and Panx1 inhibitors revert oxaliplatin-induced neuropathic pain; Panx1 inhibitors do not alter the oxaliplatin-induced mortality of cancer cells HT-29. The inhibition of Panx1 channel is suggested as a new and safe pharmacological target.