Calcium-permeable AMPA receptors trigger vesicular glutamate release from Bergmann gliosomes.

The Bergmann glia is equipped with Ca2+-permeable AMPA receptors for glutamate, indispensable for structural and functional relations between the Bergmann glia and parallel/climbing fibers-Purkinje cell synapses. To better understand roles for the Bergmann AMPA receptors, herein we investigate on gliotransmitter release and Ca2+ signals in isolated Bergmann glia processes obtained from adult rat cerebellum. We found that: 1) the rat cerebellar purified astrocyte processes (gliosomes) expressed astrocytic and Bergmann markers and exhibited negligible contamination by nerve terminals, microglia, or oligodendrocytes; 2) activation of Ca2+-permeable AMPA receptors caused Ca2+ signals in the processes, and the release of glutamate from the processes; 3) effectiveness of rose bengal, trypan blue or bafilomycin A1, indicated that activation of the AMPA receptors evoked vesicular glutamate release. Cerebellar purified nerve terminals appeared devoid of glutamate-releasing Ca2+-permeable AMPA receptors, indicating that neuronal contamination may not be the source of the signals detected. Ultrastructural analysis indicated the presence of vesicles in the cytoplasm of the processes; confocal imaging confirmed the presence of vesicular glutamate transporters in Bergmann glia processes. We conclude that: a vesicular mechanism for release of the gliotransmitter glutamate is present in mature Bergmann processes; entry of Ca2+ through the AMPA receptors located on Bergmann processes is coupled with vesicular glutamate release. The findings would add a new role for a well-known Bergmann target for glutamate (the Ca2+-permeable AMPA receptors) and a new actor (the gliotransmitter glutamate) at the cerebellar excitatory synapses onto Purkinje cells.

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 Ca(2+) -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 Mg(2+) , 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 Ca(2+) elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca(2+) -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.

Flow Synthesis and Biological Studies of an Analgesic Adamantane Derivative That Inhibits P2X7-Evoked Glutamate Release.

We report the biological evaluation of a class of adamantane derivatives, which were achieved via modified telescoped machine-assisted flow procedure. Among the series of compounds tested in this work, 5 demonstrated outstanding analgesic properties. This compound showed that its action was not mediated through direct interaction with opioid and/or cannabinoid receptors. Moreover, it did not display any significant anti-inflammatory properties. Experiments carried out on rat cerebrocortical purified synaptosomes indicated that 5 inhibits the P2X7-evoked glutamate release, which may contribute to its antinociceptive properties. Nevertheless, further experiments are ongoing to characterize the pharmacological properties and mechanism of action of this molecule.

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 Ca(2+) 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 Ca(2+)-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.

Calmidazolium selectively inhibits exocytotic glutamate release evoked by P2X7 receptor activation.

We previously observed that activation of presynaptic P2X7 receptors located on rat cerebrocortical nerve terminals induced the release of glutamate through different modes: the channel conformation allowing Ca(2+) entry triggered exocytotic release, while the receptor itself functioned as a permeation pathway for the non-exocytotic glutamate release. Considering that exocytotic and non-exocytotic glutamate release evoked by the activation of P2X7 receptors might play a role in the control of glutamatergic synapses, we investigated whether calmidazolium (which has been found to inhibit small cation currents through recombinant P2X7 receptors, but not organic molecule permeation) could distinguish between P2X7-related exocytotic and non-exocytotic modes of glutamate release. We found that calmidazolium inhibited the intrasynaptosomal Ca(2+) response to P2X7 receptor activation and the Ca(2+)-dependent exocytotic glutamate release from rat cerebrocortical nerve terminals, but was ineffective against the Ca(2+)-independent glutamate release. The P2X7 competitive antagonist A-438079 eliminated both exocytotic and non-exocytotic P2X7 receptor-evoked glutamate release. Selective inhibition of exocytotic glutamate release indicates that calmidazolium inhibits events dependent on the function of native rat P2X7 receptors as Ca(2+) channels, and suggests that it can be used as a tool to dissociate P2X7-evoked exocytotic from non-exocytotic glutamate release.