Publications by authors named "Anna Maria Pugliese"

42 Publications

New Insight into the Role of Adenosine in Demyelination, Stroke and Neuropathic Pain.

Front Pharmacol 2020 29;11:625662. Epub 2021 Jan 29.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Section of Pharmacology and Toxicology, University of Florence, Florence, Italy.

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http://dx.doi.org/10.3389/fphar.2020.625662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878385PMC
January 2021

Dexpramipexole Enhances K Currents and Inhibits Cell Excitability in the Rat Hippocampus In Vitro.

Mol Neurobiol 2021 Feb 10. Epub 2021 Feb 10.

Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Florence, Italy.

Dexpramipexole (DEX) has been described as the first-in-class F1Fo ATP synthase activator able to boost mitochondrial bioenergetics and provide neuroprotection in experimental models of ischemic brain injury. Although DEX failed in a phase III trial in patients with amyotrophic lateral sclerosis, it showed favorable safety and tolerability profiles. Recently, DEX emerged as a Nav1.8 Na channel and transient outward K (I) conductance blocker, revealing therefore an unexpected, pleiotypic pharmacodynamic profile. In this study, we performed electrophysiological experiments in vitro aimed to better characterize the impact of DEX on voltage-dependent currents and synaptic transmission in the hippocampus. By means of patch-clamp recordings on isolated hippocampal neurons, we found that DEX increases outward K currents evoked by a voltage ramp protocol. This effect is prevented by the non-selective voltage-dependent K channel (Kv) blocker TEA and by the selective small-conductance Ca-activated K (SK) channel blocker apamin. In keeping with this, extracellular field recordings from rat hippocampal slices also demonstrated that the compound inhibits synaptic transmission and CA1 neuron excitability. Overall, these data further our understanding on the pharmacodynamics of DEX and disclose an additional mechanism that could underlie its neuroprotective properties. Also, they identify DEX as a lead to develop new modulators of K conductances.
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http://dx.doi.org/10.1007/s12035-021-02300-5DOI Listing
February 2021

Oligodendrocyte precursor cell maturation: role of adenosine receptors.

Neural Regen Res 2021 Sep;16(9):1686-1692

Department of Neuroscience, Psychology, Drug Research and Child Health-Neurofarba-Division of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Oligodendrocyte-formed myelin sheaths allow fast synaptic transmission in the brain and their degeneration leads to demyelinating diseases such as multiple sclerosis. Remyelination requires the differentiation of oligodendrocyte progenitor cells into mature oligodendrocytes but, in chronic neurodegenerative disorders, remyelination fails due to adverse environment. Therefore, a strategy to prompt oligodendrocyte progenitor cell differentiation towards myelinating oligodendrocytes is required. The neuromodulator adenosine, and its receptors (A, A, A and A receptors: AR, AR, AR and AR), are crucial mediators in remyelination processes. It is known that ARs facilitate oligodendrocyte progenitor cell maturation and migration whereas the ARs initiates apoptosis in oligodendrocyte progenitor cells. Our group of research contributed to the field by demonstrating that AR and AR inhibit oligodendrocyte progenitor cell maturation by reducing voltage-dependent K currents necessary for cell differentiation. The present review summarizes the possible role of adenosine receptor ligands as potential therapeutic targets in demyelinating pathologies such as multiple sclerosis.
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http://dx.doi.org/10.4103/1673-5374.306058DOI Listing
September 2021

Photobiomodulation of Human Fibroblasts and Keratinocytes with Blue Light: Implications in Wound Healing.

Biomedicines 2021 Jan 5;9(1). Epub 2021 Jan 5.

Istituto di Fisica Applicata "Nello Carrara", Consiglio Nazionale delle Ricerche (CNR-IFAC), 50019 Florence, Italy.

In recent years, photobiomodulation (PBM) has been recognized as a physical therapy in wound management. Despite several published research papers, the mechanism underlying photobiomodulation is still not completely understood. The investigation about application of blue light to improve wound healing is a relatively new research area. Tests in selected patients evidenced a stimulation of the healing process in superficial and chronic wounds treated with a blue LED light emitting at 420 nm; a study in animal model pointed out a faster healing process in superficial wound, with an important role of fibroblasts and myofibroblasts. Here, we present a study aiming at evidencing the effects of blue light on the proliferation and metabolism in fibroblasts from healthy skin and keratinocytes. Different light doses (3.43, 6.87, 13.7, 20.6, 30.9 and 41.2 J/cm2) were used to treat the cells, evidencing inhibitory and stimulatory effects following a biphasic dose behavior. Electrophysiology was used to investigate the effects on membrane currents: healthy fibroblasts and keratinocytes showed no significant differences between treated and not treated cells. Raman spectroscopy revealed the mitochondrial Cytochrome C (Cyt C) oxidase dependence on blue light irradiation: a significant decrease in peak intensity of healthy fibroblast was evidenced, while it is less pronounced in keratinocytes. In conclusion, we observed that the blue LED light can be used to modulate metabolism and proliferation of human fibroblasts, and the effects in wound healing are particularly evident when studying the fibroblasts and keratinocytes co-cultures.
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http://dx.doi.org/10.3390/biomedicines9010041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824830PMC
January 2021

A Adenosine Receptors: When Outsiders May Become an Attractive Target to Treat Brain Ischemia or Demyelination.

Int J Mol Sci 2020 Dec 18;21(24). Epub 2020 Dec 18.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Section of Pharmacology and Toxicology, University of Florence, 50139 Florence, Italy.

Adenosine is a signaling molecule, which, by activating its receptors, acts as an important player after cerebral ischemia. Here, we review data in the literature describing AR-mediated effects in models of cerebral ischemia obtained in vivo by the occlusion of the middle cerebral artery (MCAo) or in vitro by oxygen-glucose deprivation (OGD) in hippocampal slices. Adenosine plays an apparently contradictory role in this receptor subtype depending on whether it is activated on neuro-glial cells or peripheral blood vessels and/or inflammatory cells after ischemia. Indeed, ARs participate in the early glutamate-mediated excitotoxicity responsible for neuronal and synaptic loss in the CA1 hippocampus. On the contrary, later after ischemia, the same receptors have a protective role in tissue damage and functional impairments, reducing inflammatory cell infiltration and neuroinflammation by central and/or peripheral mechanisms. Of note, demyelination following brain ischemia, or autoimmune neuroinflammatory reactions, are also profoundly affected by ARs since they are expressed by oligodendroglia where their activation inhibits cell maturation and expression of myelin-related proteins. In conclusion, data in the literature indicate the ARs as putative therapeutic targets for the still unmet treatment of stroke or demyelinating diseases.
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http://dx.doi.org/10.3390/ijms21249697DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766015PMC
December 2020

Experimental Study on Blue Light Interaction with Human Keloid-Derived Fibroblasts.

Biomedicines 2020 Dec 6;8(12). Epub 2020 Dec 6.

Istituto di Fisica Applicata "Nello Carrara", Consiglio Nazionale delle Ricerche (CNR-IFAC), 50019 Florence, Italy.

Keloids are an exuberant response to wound healing, characterized by an exaggerated synthesis of collagen, probably due to the increase of fibroblasts activity and to the reduction of their apoptosis rate: currently no standard treatments or pharmacological therapies are able to prevent keloid recurrence. To reach this goal, in recent years some physical treatments have been proposed, and among them the PhotoBioModulation therapy (PBM). This work analyses the effects of a blue LED light irradiation (410-430 nm, 0.69 W/cm power density) on human fibroblasts, isolated from both keloids and perilesional tissues. Different light doses (3.43-6.87-13.7-20.6-30.9 and 41.2 J/cm) were tested. Biochemical assays and specific staining were used to assess cell metabolism, proliferation and viability. Micro-Raman spectroscopy was used to explore direct effects of the blue LED light on the Cytochrome C (Cyt C) oxidase. We also investigated the effects of the irradiation on ionic membrane currents by patch-clamp recordings. Our results showed that the blue LED light can modulate cell metabolism and proliferation, with a dose-dependent behavior and that these effects persist at least till 48 h after treatment. Furthermore, we demonstrated that the highest fluence value can reduce cell viability 24 h after irradiation in keloid-derived fibroblasts, while the same effect is observed 48 h after treatment in perilesional fibroblasts. Electrophysiological recordings showed that the medium dose (20.6 J/cm) of blue LED light induces an enhancement of voltage-dependent outward currents elicited by a depolarizing ramp protocol. Overall, these data demonstrate the potentials that PBM shows as an innovative and minimally-invasive approach in the management of hypertrophic scars and keloids, in association with current treatments.
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http://dx.doi.org/10.3390/biomedicines8120573DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762279PMC
December 2020

Acetylcholine modulates K and Na currents in human basal forebrain cholinergic neuroblasts through an autocrine/paracrine mechanism.

J Neurochem 2020 Oct 8. Epub 2020 Oct 8.

Department of Experimental and Clinical Medicine, Section of Human Anatomy and Histology, University of Florence, Florence, Italy.

The Nucleus Basalis of Meynert (NBM) is the main source of cholinergic neurons in the basal forebrain to be crucially involved in cognitive functions and whose degeneration correlates with cognitive decline in major degenerative pathologies as Alzheimer's and Parkinson's diseases. However, knowledge concerning NBM neurons derived from human brain is very limited to date. We recently characterized a primary culture of proliferating neuroblasts isolated from the human fetal NBM (hfNBM) as immature cholinergic neurons expressing the machinery to synthetize and release acetylcholine. Here we studied in detail electrophysiological features and cholinergic effects in this cell culture by patch-clamp recordings. Our data demonstrate that atropine-blocked muscarinic receptor activation by acetylcholine or carbachol enhanced I and reduced I currents by stimulating G -coupled M2 or phospholipase C-coupled M3 receptors, respectively. Inhibition of acetylcholine esterase activity by neostigmine unveiled a spontaneous acetylcholine release from hfNBM neuroblasts that might account for an autocrine/paracrine signaling during human brain development. Present data provide the first description of cholinergic effects in human NBM neurons and point to a role of acetylcholine as an autocrine/paracrine modulator of voltage-dependent channels. Our research could be of relevance in understanding the mechanisms of cholinergic system development and functions in the human brain, either in health or disease.
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http://dx.doi.org/10.1111/jnc.15209DOI Listing
October 2020

Adenosine A receptors inhibit K currents and cell differentiation in cultured oligodendrocyte precursor cells and modulate sphingosine-1-phosphate signaling pathway.

Biochem Pharmacol 2020 07 3;177:113956. Epub 2020 Apr 3.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Section of Pharmacology and Toxicology, University of Florence, Italy.

Oligodendrocytes are the only myelinating cells in the brain and differentiate from their progenitors (OPCs) throughout adult life. However, this process fails in demyelinating pathologies. Adenosine is emerging as an important player in OPC differentiation and we recently demonstrated that adenosine A receptors inhibit cell maturation by reducing voltage-dependent K currents. No data are available to date about the A receptor (AR) subtype. The bioactive lipid mediator sphingosine-1-phosphate (S1P) and its receptors (S1P) are also crucial modulators of OPC development. An interaction between this pathway and the AR is reported in peripheral cells. We studied the role of ARs in modulating K currents and cell differentiation in OPC cultures and we investigated a possible interplay with S1P signaling. Our data indicate that the AR agonist BAY60-6583 and its new analogue P453 inhibit K currents in cultured OPC and the effect was prevented by the AR antagonist MRS1706, by K channel blockers and was differently modulated by the S1P analogue FTY720-P. An acute (10 min) exposure of OPCs to BAY60-6583 also increased the phosphorylated form of sphingosine kinase 1 (SphK1). A chronic (7 days) treatment with the same agonist decreased OPC differentiation whereas SphK1/2 inhibition exerted the opposite effect. Furthermore, AR was overexpressed during OPC differentiation, an effect prevented by the pan SphK1/2 inhibitor VPC69047. Finally, AR silenced cells showed increased cell maturation, decreased SphK1 expression and enhanced S1P lyase levels. We conclude that ARs inhibit K currents and cell differentiation and positively modulate S1P synthesis in cultured OPCs.
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http://dx.doi.org/10.1016/j.bcp.2020.113956DOI Listing
July 2020

Dexpramipexole blocks Nav1.8 sodium channels and provides analgesia in multiple nociceptive and neuropathic pain models.

Pain 2020 04;161(4):831-841

Section of Clinical Pharmacology and Oncology, Department of Health Sciences, University of Florence, Florence, Italy.

Selective targeting of sodium channel subtypes Nav1.7, Nav1.8, and Nav1.9, preferentially expressed by peripheral nociceptors, represents a unique opportunity to develop analgesics devoid of central side effects. Several compounds that target Nav1.7 and Nav1.8 with different degrees of selectivity have been developed and are currently being tested in clinical trials for multiple pain indications. Among these chemicals, benzothiazole-like compounds emerged as potent sodium channel blockers. We evaluated the effects of dexpramipexole, a benzothiazole-bearing drug with pleiotypic neuroactive properties and a good safety profile in humans, on sodium conductances of dorsal root ganglia neurons, as well as in multiple nociceptive and neuropathic pain models. Dexpramipexole blocks TTX-resistant sodium conductances in cultured rat dorsal root ganglion neurons with an IC50 of 294.4 nM, suggesting selectivity towards Nav1.8. In keeping with this, dexpramipexole does not affect sodium currents in dorsal root ganglion neurons from Nav1.8 null mice and acquires binding pose predicted to overlap that of the Nav1.8 channel-selective blocker A-8034637. The drug provides analgesia when parenterally, orally, or topically applied in inflammatory and visceral mouse pain models, as well as in mice affected by neuropathic pain induced by oxaliplatin, nerve constriction, or diabetes. Pain reduction in mice occurs at doses consistent with those adopted in clinical trials. The present findings confirm the relevance of selective targeting of peripheral Nav1.8 channels to pain therapy. In light of the excellent tolerability of dexpramipexole in humans, our results support its translational potential for treatment of pain.
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http://dx.doi.org/10.1097/j.pain.0000000000001774DOI Listing
April 2020

Functional characterization of a novel adenosine A receptor agonist on short-term plasticity and synaptic inhibition during oxygen and glucose deprivation in the rat CA1 hippocampus.

Brain Res Bull 2019 09 24;151:174-180. Epub 2019 May 24.

Department of Neuroscience, Psychology, Drug Research and Child Health, NEUROFARBA, Section of Pharmacology and Toxicology, University of Florence, Florence, Italy. Electronic address:

Adenosine is an endogenous neuromodulator exerting its biological functions via four receptor subtypes, A, A, A, and A. A receptors (ARs) are expressed at hippocampal level where they are known to inhibit paired pulse facilitation (PPF), whose reduction reflects an increase in presynaptic glutamate release. The effect of ARs on PPF is known to be sensitive not only to AR blockade but also to the AR antagonist DPCPX, indicating that it involves AR activation. In this study we provide the first functional characterization of the newly synthesized non-nucleoside like AR agonist P453, belonging to the amino-3,5-dicyanopyridine series. By extracellular electrophysiological recordings, we demonstrated that P453 mimicked the effect of the prototypical AR agonist BAY60-6583 in decreasing PPF at Schaffer collateral-CA1 synapses in rat acute hippocampal slices. This effect was prevented by two different AR antagonists, PSB603 and MRS1754, and by the AR antagonist DPCPX. We also investigated the functional role of AR during a 2 min of oxygen and glucose deprivation (OGD) insult, known to produce a reversible fEPSP inhibition due to adenosine AR activation. We found that P453 and BAY60-6583 significantly delayed the onset of fEPSP reduction induced by OGD and the effect was blocked by PSB603. We conclude that P453 is a functional AR agonist whose activation decreases PPF by increasing glutamate release at presynaptic terminals and delays AR-mediated fEPSP inhibition during a 2-minute OGD insult.
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http://dx.doi.org/10.1016/j.brainresbull.2019.05.018DOI Listing
September 2019

Adenosine A3 receptor activation inhibits pronociceptive N-type Ca2+ currents and cell excitability in dorsal root ganglion neurons.

Pain 2019 05;160(5):1103-1118

Division of Pharmacology and Toxicology, Department of NEUROFARBA, University of Florence, Italy.

Recently, studies have focused on the antihyperalgesic activity of the A3 adenosine receptor (A3AR) in several chronic pain models, but the cellular and molecular basis of this effect is still unknown. Here, we investigated the expression and functional effects of A3AR on the excitability of small- to medium-sized, capsaicin-sensitive, dorsal root ganglion (DRG) neurons isolated from 3- to 4-week-old rats. Real-time quantitative polymerase chain reaction experiments and immunofluorescence analysis revealed A3AR expression in DRG neurons. Patch-clamp experiments demonstrated that 2 distinct A3AR agonists, Cl-IB-MECA and the highly selective MRS5980, inhibited Ca-activated K (KCa) currents evoked by a voltage-ramp protocol. This effect was dependent on a reduction in Ca influx via N-type voltage-dependent Ca channels, as Cl-IB-MECA-induced inhibition was sensitive to the N-type blocker PD173212 but not to the L-type blocker, lacidipine. The endogenous agonist adenosine also reduced N-type Ca currents, and its effect was inhibited by 56% in the presence of A3AR antagonist MRS1523, demonstrating that the majority of adenosine's effect is mediated by this receptor subtype. Current-clamp recordings demonstrated that neuronal firing of rat DRG neurons was also significantly reduced by A3AR activation in a MRS1523-sensitive but PD173212-insensitive manner. Intracellular Ca measurements confirmed the inhibitory role of A3AR on DRG neuronal firing. We conclude that pain-relieving effects observed on A3AR activation could be mediated through N-type Ca channel block and action potential inhibition as independent mechanisms in isolated rat DRG neurons. These findings support A3AR-based therapy as a viable approach to alleviate pain in different pathologies.
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http://dx.doi.org/10.1097/j.pain.0000000000001488DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6669900PMC
May 2019

Dexpramipexole enhances hippocampal synaptic plasticity and memory in the rat.

Neuropharmacology 2018 12 3;143:306-316. Epub 2018 Oct 3.

Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Italy.

Even though pharmacological approaches able to counteract age-dependent cognitive impairment have been highly investigated, drugs improving cognition and memory are still an unmet need. It has been hypothesized that sustaining energy dynamics within the aged hippocampus can boost memory storage by sustaining synaptic functioning and long term potentiation (LTP). Dexpramipexole (DEX) is the first-in-class compound able to sustain neuronal bioenergetics by interacting with mitochondrial F1Fo-ATP synthase. In the present study, for the first time we evaluated the effects of DEX on synaptic fatigue, LTP induction, learning and memory retention. We report that DEX improved LTP maintenance in CA1 neurons of acute hippocampal slices from aged but not young rats. However, we found no evidence that DEX counteracted two classic parameters of synaptic fatigue such as fEPSP reduction or the train area during the high frequency stimulation adopted to induce LTP. Interestingly, patch-clamp recordings in rat hippocampal neurons revealed that DEX dose-dependently inhibited (IC 814 nM) the I current, a rapidly-inactivating K current that negatively regulates neuronal excitability as well as cognition and memory processes. In keeping with this, DEX counteracted both scopolamine-induced spatial memory loss in rats challenged in Morris Water Maze test and memory retention in rats undergoing Novel Object Recognition. Overall, the present study discloses the ability of DEX to boost hippocampal synaptic plasticity, learning and memory. In light of the good safety profile of DEX in humans, our findings may have a realistic translational potential to treatment of cognitive disorders.
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http://dx.doi.org/10.1016/j.neuropharm.2018.10.003DOI Listing
December 2018

Imidazo[1,2-a]pyrazin-8-amine core for the design of new adenosine receptor antagonists: Structural exploration to target the A and A subtypes.

Eur J Med Chem 2017 Jan 26;125:611-628. Epub 2016 Sep 26.

Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sez. Farmaceutica e Nutraceutica, Universita' degli Studi di Firenze, Via Ugo Schiff 6, 50019, Sesto Fiorentino (FI), Italy.

The imidazo[1,2-a]pyrazine ring system has been chosen as a new decorable core skeleton for the design of novel adenosine receptor (AR) antagonists targeting either the human (h) A or the hA receptor subtype. The N-(hetero)arylcarboxyamido substituted compounds 4-14 and 21-30, bearing a 6-phenyl moiety or not, respectively, show good hA receptor affinity and selectivity versus the other ARs. In contrast, the 8-amino-6-(hetero)aryl substituted derivatives designed for targeting the hA receptor subtype (compounds 31-38) and also the 6-phenyl analogues 18-20 do not bind the hA AR, or show hA or balanced hA/hA AR affinity in the micromolar range. Molecular docking of the new hA antagonists was carried out to depict their hypothetical binding mode to our refined model of the hA receptor. Some derivatives were evaluated for their fluorescent potentiality and showed some fluorescent emission properties. One of the most active hA antagonists herein reported, i.e. the 2,6-diphenyl-8-(3-pyridoylamino)imidazo[1,2-a]pyrazine 29, tested in a rat model of cerebral ischemia, delayed the occurrence of anoxic depolarization caused by oxygen and glucose deprivation in the hippocampus and allowed disrupted synaptic activity to recover.
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http://dx.doi.org/10.1016/j.ejmech.2016.09.076DOI Listing
January 2017

Purinergic signalling in brain ischemia.

Neuropharmacology 2016 05 12;104:105-30. Epub 2015 Nov 12.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy.

Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia a primary damage due to the early massive increase of extracellular glutamate is followed by activation of resident immune cells, i.e microglia, and production or activation of inflammation mediators. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. Extracellular concentrations of ATP and adenosine in the brain increase dramatically during ischemia in concentrations able to stimulate their respective specific P2 and P1 receptors. Both ATP P2 and adenosine P1 receptor subtypes exert important roles in ischemia. Although adenosine exerts a clear neuroprotective effect through A1 receptors during ischemia, the use of selective A1 agonists is hampered by undesirable peripheral effects. Evidence up to now in literature indicate that A2A receptor antagonists provide protection centrally by reducing excitotoxicity, while agonists at A2A (and possibly also A2B) and A3 receptors provide protection by controlling massive infiltration and neuroinflammation in the hours and days after brain ischemia. Among P2X receptors most evidence indicate that P2X7 receptor contribute to the damage induced by the ischemic insult due to intracellular Ca(2+) loading in central cells and facilitation of glutamate release. Antagonism of P2X7 receptors might represent a new treatment to attenuate brain damage and to promote proliferation and maturation of brain immature resident cells that can promote tissue repair following cerebral ischemia. Among P2Y receptors, antagonists of P2Y12 receptors are of value because of their antiplatelet activity and possibly because of additional anti-inflammatory effects. Moreover strategies that modify adenosine or ATP concentrations at injury sites might be of value to limit damage after ischemia. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.
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http://dx.doi.org/10.1016/j.neuropharm.2015.11.007DOI Listing
May 2016

Role of adenosine in oligodendrocyte precursor maturation.

Front Cell Neurosci 2015 24;9:155. Epub 2015 Apr 24.

Department NEUROFARBA, Division of Pharmacology and Toxicology, University of Florence Florence, Italy.

Differentiation and maturation of oligodendroglial cells are postnatal processes that involve specific morphological changes correlated with the expression of stage-specific surface antigens and functional voltage-gated ion channels. A small fraction of oligodendrocyte progenitor cells (OPCs) generated during development are maintained in an immature and slowly proliferative or quiescent state in the adult central nervous system (CNS) representing an endogenous reservoir of immature cells. Adenosine receptors are expressed by OPCs and a key role of adenosine in oligodendrocyte maturation has been recently recognized. As evaluated on OPC cultures, adenosine, by stimulating A1 receptors, promotes oligodendrocyte maturation and inhibits their proliferation; on the contrary, by stimulating A2A receptors, it inhibits oligodendrocyte maturation. A1 and A2A receptor-mediated effects are related to opposite modifications of outward delayed rectifying membrane K(+) currents (IK) that are involved in the regulation of oligodendrocyte differentiation. Brain A1 and A2A receptors might represent new molecular targets for drugs useful in demyelinating pathologies, such as multiple sclerosis (MS), stroke and brain trauma.
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http://dx.doi.org/10.3389/fncel.2015.00155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408841PMC
May 2015

The selective antagonism of P2X7 and P2Y1 receptors prevents synaptic failure and affects cell proliferation induced by oxygen and glucose deprivation in rat dentate gyrus.

PLoS One 2014 19;9(12):e115273. Epub 2014 Dec 19.

Dept. of Neuroscience, Psychology, Drug Research and Child Health, NEUROFARBA, Division of Pharmacology and Toxicology, University of Florence, Florence, Italy.

Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X7 and P2Y1 receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y1 receptor) and BBG (selective antagonist of P2X7 receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2'-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU+ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU+ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y1 antagonist reduced the number of BrdU+ cells at this time. The data demonstrate that P2X7 and P2Y1 activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y1 receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115273PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4272279PMC
December 2015

The neuron-astrocyte-microglia triad in a rat model of chronic cerebral hypoperfusion: protective effect of dipyridamole.

Front Aging Neurosci 2014 27;6:322. Epub 2014 Nov 27.

Section of Pharmacology and Clinical Oncology, Department of Health Sciences, University of Florence Florence, Italy.

Chronic cerebral hypoperfusion during aging may cause progressive neurodegeneration as ischemic conditions persist. Proper functioning of the interplay between neurons and glia is fundamental for the functional organization of the brain. The aim of our research was to study the pathophysiological mechanisms, and particularly the derangement of the interplay between neurons and astrocytes-microglia with the formation of "triads," in a model of chronic cerebral hypoperfusion induced by the two-vessel occlusion (2VO) in adult Wistar rats (n = 15). The protective effect of dipyridamole given during the early phases after 2VO (4 mg/kg/day i.v., the first 7 days after 2VO) was verified (n = 15). Sham-operated rats (n = 15) were used as controls. Immunofluorescent triple staining of neurons (NeuN), astrocytes (GFAP), and microglia (IBA1) was performed 90 days after 2VO. We found significantly higher amount of "ectopic" neurons, neuronal debris and apoptotic neurons in CA1 Str. Radiatum and Str. Pyramidale of 2VO rats. In CA1 Str. Radiatum of 2VO rats the amount of astrocytes (cells/mm(2)) did not increase. In some instances several astrocytes surrounded ectopic neurons and formed a "micro scar" around them. Astrocyte branches could infiltrate the cell body of ectopic neurons, and, together with activated microglia cells formed the "triads." In the triad, significantly more numerous in CA1 Str. Radiatum of 2VO than in sham rats, astrocytes and microglia cooperated in the phagocytosis of ectopic neurons. These events might be common mechanisms underlying many neurodegenerative processes. The frequency to which they appear might depend upon, or might be the cause of, the burden and severity of neurodegeneration. Dypiridamole significantly reverted all the above described events. The protective effect of chronic administration of dipyridamole might be a consequence of its vasodilatory, antioxidant and anti-inflammatory role during the early phases after 2VO.
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http://dx.doi.org/10.3389/fnagi.2014.00322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4245920PMC
December 2014

Oleuropein aglycone protects against pyroglutamylated-3 amyloid-ß toxicity: biochemical, epigenetic and functional correlates.

Neurobiol Aging 2015 Feb 6;36(2):648-63. Epub 2014 Sep 6.

Department of Neuroscience, Psychology, Drug Research and Child Health, Division of Pharmacology and Toxicology, University of Florence, Florence, Italy. Electronic address:

Amyloid-ß (Aß) fragments, oligomeric Aß aggregates, and pyroglutamylated-Aß peptides, as well as epigenetic mechanisms and autophagy dysfunction all appear to contribute in various ways to Alzheimer's disease progression. We previously showed that dietary supplementation of oleuropein aglycone, a natural phenol abundant in the extra virgin olive oil, can be protective by reducing Aß42 deposits in the brain of young and middle-aged TgCRND8 mice. Here, we extended our study to aged TgCRND8 mice showing increased pE3-Aß in the brain deposits. We report that oleuropein aglycone is active against glutaminylcyclase-catalyzed pE3-Aß generation reducing enzyme expression and interferes both with Aß42 and pE3-Aß aggregation. Moreover, the phenol astonishingly activates neuronal autophagy even in mice at advanced stage of pathology, where it increases histone 3 and 4 acetylation, which matches both a decrease of histone deacetylase 2 expression and a significant improvement of synaptic function. The occurrence of these functional, epigenetic, and histopathologic beneficial effects even at a late stage of the pathology suggests that the phenol could be beneficial at the therapeutic, in addition to the prevention, level.
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http://dx.doi.org/10.1016/j.neurobiolaging.2014.08.029DOI Listing
February 2015

Adenosine receptors in cerebral ischemia.

Int Rev Neurobiol 2014 ;119:309-48

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Division of Pharmacology and Toxicology, University of Florence, 50139 Florence, Italy.

Ischemic stroke is a complex pathology characterized by a sequence of events that evolve over time and space. It is the second leading cause of death and the main cause of adult long-term disability in developed countries. At the moment, there is no promising pharmacotherapy for acute ischemic stroke. Adenosine receptors (A1, A2A, A2B, A3) are important targets for therapeutic implementation in the treatment of stroke because extracellular adenosine concentrations increase dramatically soon after ischemia. Adenosine receptors located both on central nervous system cells and on immune blood cells exert important roles during ischemia. The neuroprotective role of adenosine through A1 receptor subtype during ischemia is accepted, but the use of selective A1 agonists is hampered by undesirable side effects such as sedation, bradycardia, and hypotension. Recently, the A2A receptor subtype emerged as a potential therapeutic attractive target in ischemia. Evidence suggests that A2A receptor has dual role: in a first phase of ischemia, it potentiates excitotoxicity, while hours and days after ischemia, A2A receptors on immune blood cells potentiate cell adhesion mechanisms and infiltration in the ischemic parenchyma. Consistently, the use of A2A receptor agonists/antagonists (administered at doses that do not modify blood pressure and heart rate) should be carefully evaluated in function of time after ischemia. Although much is still to be known about the role of A2B and A3 receptor subtypes in brain ischemia, most consistent information indicates their role in regulation of immunosuppression and inflammation.
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http://dx.doi.org/10.1016/B978-0-12-801022-8.00013-1DOI Listing
May 2015

Adenosine A2A receptors modulate acute injury and neuroinflammation in brain ischemia.

Mediators Inflamm 2014 5;2014:805198. Epub 2014 Aug 5.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.

The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A(2A) receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A(2A) receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A(2A) receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A(2A) receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A(2A) receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A(2A) receptor agonists a wide therapeutic time-window of hours and even days after stroke.
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http://dx.doi.org/10.1155/2014/805198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4138795PMC
May 2015

Hippocampal long term memory: effect of the cholinergic system on local protein synthesis.

Neurobiol Learn Mem 2013 Nov 27;106:246-57. Epub 2013 Sep 27.

Department of Health Sciences, Section of Pharmacology and Clinical Oncology, University of Florence, Viale Pieraccini 6, 50139 Firenze, Italy.

The present study was aimed at establishing a link between the cholinergic system and the pathway of mTOR and its downstream effector p70S6K, likely actors in long term memory encoding. We performed in vivo behavioral experiments using the step down inhibitory avoidance test (IA) in adult Wistar rats to evaluate memory formation under different conditions, and immunohistochemistry on hippocampal slices to evaluate the level and the time-course of mTOR and p70S6K activation. We also examined the effect of RAPA, inhibitor of mTORC1 formation, and of the acetylcholine (ACh) muscarinic receptor antagonist scopolamine (SCOP) or ACh nicotinic receptor antagonist mecamylamine (MECA) on short and long term memory formation and on the functionality of the mTOR pathway. Acquisition test was performed 30 min after i.c.v. injection of RAPA, a time sufficient for the drug to diffuse to CA1 pyramidal neurons, as demonstrated by MALDI-TOF-TOF imaging. Recall test was performed 1 h, 4 h or 24 h after acquisition. To confirm our results we performed in vitro experiments on live hippocampal slices: we evaluated whether stimulation of the cholinergic system with the cholinergic receptor agonist carbachol (CCh) activated the mTOR pathway and whether the administration of the above-mentioned antagonists together with CCh could revert this activation. We found that (1) mTOR and p70S6K activation in the hippocampus were involved in long term memory formation; (2) RAPA administration caused inhibition of mTOR activation at 1 h and 4 h and of p70S6K activation at 4 h, and long term memory impairment at 24 h after acquisition; (3) scopolamine treatment caused short but not long term memory impairment with an early increase of mTOR/p70S6K activation at 1 h followed by stabilization at longer times; (4) mecamylamine plus scopolamine treatment caused short term memory impairment at 1 h and 4 h and reduced the scopolamine-induced increase of mTOR/p70S6K activation at 1 h and 4 h; (5) mecamylamine plus scopolamine treatment did not impair long term memory formation; (6) in vitro treatment with carbachol activated mTOR and p70S6K and this effect was blocked by scopolamine and mecamylamine. Taken together our data reinforce the idea that distinct molecular mechanisms are at the basis of the two different forms of memory and are in accordance with data presented by other groups that there exist molecular mechanisms that underlie short term memory, others that underlie long term memories, but some mechanisms are involved in both.
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http://dx.doi.org/10.1016/j.nlm.2013.09.013DOI Listing
November 2013

Anticonvulsant effect of AMP by direct activation of adenosine A1 receptor.

Exp Neurol 2013 Dec 19;250:189-93. Epub 2013 Sep 19.

Department of Health Sciences, Section of Clinical Pharmacology and Oncology, University of Florence, Italy. Electronic address:

Purinergic neurotransmission mediated by adenosine (Ado) type 1 receptors (A1Rs) plays pivotal roles in negative modulation of epileptic seizures, and Ado is thought to be a key endogenous anticonvulsant. Recent evidence, however, indicates that AMP, the metabolic precursor of Ado, also activate A1Rs. Here, we evaluated the antiepileptic effects of AMP adopting in vitro and in vivo models of epilepsy. We report that AMP reversed the increase in population spike (PS) amplitude and the decrease in PS latency induced by a Mg(2+)-free extracellular solution in CA1 neurons of mouse hippocampal slices. The AMP effects were inhibited by the A1R antagonist DPCPX, but not prevented by inhibiting conversion of AMP into Ado, indicating that AMP inhibited per se sustained hippocampal excitatory neurotransmission by directly activating A1Rs. AMP also reduced seizure severity and mortality in a model of audiogenic convulsion. Of note, the anticonvulsant effects of AMP were potentiated by preventing its conversion into Ado and inhibited by DPCPX. When tested in a model of kainate-induced seizure, AMP prolonged latency of convulsions but had no effects on seizure severity and mortality. Data provide the first evidence that AMP is an endogenous anticonvulsant acting at A1Rs.
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http://dx.doi.org/10.1016/j.expneurol.2013.09.010DOI Listing
December 2013

Adenosine A₂A receptors inhibit delayed rectifier potassium currents and cell differentiation in primary purified oligodendrocyte cultures.

Neuropharmacology 2013 Oct 13;73:301-10. Epub 2013 Jun 13.

Department of Neuroscience, Psychology, Drug Research and Child Health (NEUROFARBA), Division of Pharmacology and Toxicology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.

Oligodendrocyte progenitor cells (OPCs) are a population of cycling cells which persist in the adult central nervous system (CNS) where, under opportune stimuli, they differentiate into mature myelinating oligodendrocytes. Adenosine A(2A) receptors are Gs-coupled P1 purinergic receptors which are widely distributed throughout the CNS. It has been demonstrated that OPCs express A(2A) receptors, but their functional role in these cells remains elusive. Oligodendrocytes express distinct voltage-gated ion channels depending on their maturation. Here, by electrophysiological recordings coupled with immunocytochemical labeling, we studied the effects of adenosine A(2A) receptors on membrane currents and differentiation of purified primary OPCs isolated from the rat cortex. We found that the selective A(2A) agonist, CGS21680, inhibits sustained, delayed rectifier, K(+) currents (I(K)) without modifying transient (I(A)) conductances. The effect was observed in all cells tested, independently from time in culture. CGS21680 inhibition of I(K) current was concentration-dependent (10-200 nM) and blocked in the presence of the selective A(2A) antagonist SCH58261 (100 nM). It is known that I(K) currents play an important role during OPC development since their block decreases cell proliferation and differentiation. In light of these data, our further aim was to investigate whether A(2A) receptors modulate these processes. CGS21680, applied at 100 nM in the culture medium of oligodendrocyte cultures, inhibits OPC differentiation (an effect prevented by SCH58261) without affecting cell proliferation. Data demonstrate that cultured OPCs express functional A(2A) receptors whose activation negatively modulate I(K) currents. We propose that, by this mechanism, A(2A) adenosine receptors inhibit OPC differentiation.
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http://dx.doi.org/10.1016/j.neuropharm.2013.05.035DOI Listing
October 2013

UDP-glucose enhances outward K(+) currents necessary for cell differentiation and stimulates cell migration by activating the GPR17 receptor in oligodendrocyte precursors.

Glia 2013 Jul 2;61(7):1155-71. Epub 2013 May 2.

Divi Department of Neuroscience, Psychology, Drug Research and Child Health (NeuroFarBa), University of Florence, Viale Pieraccini, 6, 50139 Florence, Italy.

In the developing and mature central nervous system, NG2 expressing cells comprise a population of cycling oligodendrocyte progenitor cells (OPCs) that differentiate into mature, myelinating oligodendrocytes (OLGs). OPCs are also characterized by high motility and respond to injury by migrating into the lesioned area to support remyelination. K(+) currents in OPCs are developmentally regulated during differentiation. However, the mechanisms regulating these currents at different stages of oligodendrocyte lineage are poorly understood. Here we show that, in cultured primary OPCs, the purinergic G-protein coupled receptor GPR17, that has recently emerged as a key player in oligodendrogliogenesis, crucially regulates K(+) currents. Specifically, receptor stimulation by its agonist UDP-glucose enhances delayed rectifier K(+) currents without affecting transient K(+) conductances. This effect was observed in a subpopulation of OPCs and immature pre-OLGs whereas it was absent in mature OLGs, in line with GPR17 expression, that peaks at intermediate phases of oligodendrocyte differentiation and is thereafter downregulated to allow terminal maturation. The effect of UDP-glucose on K(+) currents is concentration-dependent, blocked by the GPR17 antagonists MRS2179 and cangrelor, and sensitive to the K(+) channel blocker tetraethyl-ammonium, which also inhibits oligodendrocyte maturation. We propose that stimulation of K(+) currents is responsible for GPR17-induced oligodendrocyte differentiation. Moreover, we demonstrate, for the first time, that GPR17 activation stimulates OPC migration, suggesting an important role for this receptor after brain injury. Our data indicate that modulation of GPR17 may represent a strategy to potentiate the post-traumatic response of OPCs under demyelinating conditions, such as multiple sclerosis, stroke, and brain trauma.
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http://dx.doi.org/10.1002/glia.22506DOI Listing
July 2013

Amyloid-β oligomer synaptotoxicity is mimicked by oligomers of the model protein HypF-N.

Neurobiol Aging 2013 Sep 18;34(9):2100-9. Epub 2013 Apr 18.

Department of Biomedical, Experimental and Clinical Sciences, University of Florence, Florence, Italy.

Protein misfolded oligomers are thought to be the primary pathogenic species in many protein deposition diseases. Oligomers by the amyloid-β peptide play a central role in Alzheimer's disease pathogenesis, being implicated in synaptic dysfunction. Here we show that the oligomers formed by a protein that has no link with human disease, namely the N-terminal domain of HypF from Escherichia coli (HypF-N), are also synaptotoxic. HypF-N oligomers were found to (i) colocalize with post-synaptic densities in primary rat hippocampal neurons; (ii) induce impairment of long-term potentiation in rat hippocampal slices; and (iii) impair spatial learning of rats in the Morris Water Maze test. By contrast, the native protein and control nontoxic oligomers had none of such effects. These results raise the importance of using HypF-N oligomers as a valid tool to investigate the pathogenesis of Alzheimer's disease, with advantages over other systems for their stability, reproducibility, and costs. The results also suggest that, in the context of a compromised protein homeostasis resulting from aggregation of the amyloid β peptide, a number of oligomeric species sharing common synaptotoxic activity can arise and cooperate in the pathogenesis of the disease.
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http://dx.doi.org/10.1016/j.neurobiolaging.2013.03.020DOI Listing
September 2013

Effects of oxygen and glucose deprivation on synaptic transmission in rat dentate gyrus: role of A2A adenosine receptors.

Neuropharmacology 2013 Apr 19;67:511-20. Epub 2012 Dec 19.

Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.

The hippocampus is comprised of two distinct subfields that show different responses to hypoxic-ischemic brain injury: the CA1 region is particularly susceptible whereas the dentate gyrus (DG) is quite resistant. Our aim was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of A(2A) adenosine receptor antagonism to recovery of synaptic activity after OGD. Extracellular recordings of field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG in brain slices prepared from male Wistar rats were used. A 9-min OGD is needed in the DG to always induce the appearance of anoxic depolarization (AD) and the irreversible block of synaptic activity, as recorded up to 24 h from the end of the insult, whereas only 7-min OGD is required in the CA1 region. Selective antagonism of A(2A) adenosine receptors by ZM241385 significantly prevents or delays the appearance of AD and protects from the irreversible block of neurotransmission induced by 9-min OGD in the DG. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone of DG in slices prepared from 5-bromo-2'-deoxyuridine (BrdU) treated rats was investigated. Slices were further incubated with an immature neuronal marker, doublecortin (DCX). The number of BrdU(+) cells was significantly decreased 6 h after 9-min OGD and this effect was antagonized by ZM241385. After 24 h from the end of 9-min OGD, the number of BrdU(+) cells returned to that found before OGD and increased arborization of tertiary dendrites of DCX(+) cells was observed. The adenosine A(2A) antagonist ZM241385 protects from synaptic failure and from decreased proliferation of immature neuronal cells at a precocious time after OGD.
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http://dx.doi.org/10.1016/j.neuropharm.2012.12.002DOI Listing
April 2013

Neurological basis of AMP-dependent thermoregulation and its relevance to central and peripheral hyperthermia.

J Cereb Blood Flow Metab 2013 Feb 24;33(2):183-90. Epub 2012 Oct 24.

Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy.

Therapeutic hypothermia is of relevance to treatment of increased body temperature and brain injury, but drugs inducing selective, rapid, and safe cooling in humans are not available. Here, we show that injections of adenosine 5'-monophosphate (AMP), an endogenous nucleotide, promptly triggers hypothermia in mice by directly activating adenosine A1 receptors (A1R) within the preoptic area (POA) of the hypothalamus. Inhibition of constitutive degradation of brain extracellular AMP by targeting ecto 5'-nucleotidase, also suffices to prompt hypothermia in rodents. Accordingly, sensitivity of mice and rats to the hypothermic effect of AMP is inversely related to their hypothalamic 5'-nucleotidase activity. Single-cell electrophysiological recording indicates that AMP reduces spontaneous firing activity of temperature-insensitive neurons of the mouse POA, thereby retuning the hypothalamic thermoregulatory set point towards lower temperatures. Adenosine 5'-monophosphate also suppresses prostaglandin E2-induced fever in mice, having no effects on peripheral hyperthermia triggered by dioxymetamphetamine (ecstasy) overdose. Together, data disclose the role of AMP, 5'-nucleotidase, and A1R in hypothalamic thermoregulation, as well and their therapeutic relevance to treatment of febrile illness.
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http://dx.doi.org/10.1038/jcbfm.2012.157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3564191PMC
February 2013

3-Hydroxy-1H-quinazoline-2,4-dione derivatives as new antagonists at ionotropic glutamate receptors: molecular modeling and pharmacological studies.

Eur J Med Chem 2012 Aug 4;54:470-82. Epub 2012 Jun 4.

Dipartimento di Scienze Farmaceutiche, Laboratorio di Progettazione, Sintesi e Studio di Eterocicli Biologicamente Attivi, Universita' di Firenze, Polo Scientifico, Via Ugo Schiff 6, 50019 Sesto Fiorentino, FI, Italy.

Based on our 3-hydroxy-7-chloroquinazoline-2,4-dione derivatives, previously reported as antagonists at ionotropic glutamate receptors, we synthesized new 3-hydroxyquinazoline-2,4-diones bearing a trifluoromethyl group at the 7-position and different groups at position 6. Glycine/NMDA, AMPA and kainate receptor binding data showed that the 7-trifluoromethyl residue increased AMPA and kainate receptor affinity and selectivity, with respect to the 7-chlorine atom. Among the probed 6-substituents, the 6-(1,2,4-triazol-4-yl) group (compound 8) was the most advantageous for AMPA receptor affinity and selectivity. Derivative 8 demonstrated to be effective in decreasing neuronal damage produced by oxygen and glucose deprivation in organotypic rat hippocampal slices and also showed anticonvulsant effects in pentylenetetrazole-induced convulsions. The previously reported kainate receptor antagonist 6-(2-carboxybenzoyl)-amino-7-chloro-3-hydroxyquinazoline-2,4-dione 3 prevented the failure of neurotransmission induced by oxygen and glucose deprivation in the CA1 region of rat hippocampal slices.
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http://dx.doi.org/10.1016/j.ejmech.2012.05.036DOI Listing
August 2012

P2 receptor antagonists prevent synaptic failure and extracellular signal-regulated kinase 1/2 activation induced by oxygen and glucose deprivation in rat CA1 hippocampus in vitro.

Eur J Neurosci 2011 Jun 1;33(12):2203-15. Epub 2011 Apr 1.

Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy.

To investigate the role of purinergic P2 receptors under ischemia, we studied the effect of P2 receptor antagonists on synaptic transmission and mitogen-activated protein kinase (MAPK) activation under oxygen and glucose deprivation (OGD) in rat hippocampal slices. The effect of the P2 antagonists pyridoxalphosphate-6-azophenyl-2',4'-disulfonate (PPADS, unselective, 30 μm), N( 6) -methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, selective for P2Y(1) receptor, 10 μm), Brilliant Blue G (BBG, selective for P2X(7) receptor, 1 μm), and 5-[[[(3-phenoxyphenyl)methyl][(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl]-1,2,4-benzenetricarboxylic acid (A-317491, selective for P2X(3) receptor, 10 μm), and of the newly synthesized P2X(3) receptor antagonists 2-amino-9-(5-iodo-2-isopropyl-4-methoxybenzyl)adenine (PX21, 1 μm) and 2-amino-9-(5-iodo-2-isopropyl-4-methoxybenzyl)-N( 6)-methyladenine (PX24, 1 μm), on the depression of field excitatory postsynaptic potentials (fEPSPs) and anoxic depolarization (AD) elicited by 7 min of OGD were evaluated. All antagonists significantly prevented these effects. The extent of CA1 cell injury was assessed 3 h after the end of 7 min of OGD by propidium iodide staining. Substantial CA1 pyramidal neuronal damage, detected in untreated slices exposed to OGD injury, was significantly prevented by PPADS (30 μm), MRS2179 (10 μm), and BBG (1 μm). Western blot analysis showed that, 10 min after the end of the 7 min of OGD, extracellular signal-regulated kinase (ERK)1/2 MAPK activation was significantly increased. MRS2179, BBG, PPADS and A-317491 significantly counteracted ERK1/2 activation. Hippocampal slices incubated with the ERK1/2 inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126, 10 μm) and α-[amino[(4-aminophenyl)thio]methylene]-2-(trifluoromethyl) benzeneacetonitrile (SL327, 10 μm) showed significant fEPSP recovery after OGD and delayed AD, supporting the involvement of ERK1/2 in neuronal damage induced by OGD. These results indicate that subtypes of hippocampal P2 purinergic receptors have a harmful effect on neurotransmission in the CA1 hippocampus by participating in AD appearance and activation of ERK1/2.
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http://dx.doi.org/10.1111/j.1460-9568.2011.07667.xDOI Listing
June 2011

Functional characterization of two isoforms of the P2Y-like receptor GPR17: [35S]GTPgammaS binding and electrophysiological studies in 1321N1 cells.

Am J Physiol Cell Physiol 2009 Oct 22;297(4):C1028-40. Epub 2009 Jul 22.

Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy.

The previously "orphan" G protein-coupled receptor GPR17 is structurally related to both P2Y nucleotide receptors and to receptors for cysteinyl leukotrienes. Genomic analysis revealed two putative open reading frames encoding for a "short" and a "long" receptor isoform of 339- and 367-amino acids, respectively, with the latter displaying a 28-amino acid longer NH(2) terminus. The short isoform has been recently "deorphanized," revealing dual responses to uracil nucleotides and cysteinyl leukotrienes. No information regarding the ligand specificity, tissue distribution, or pathophysiological roles of the long receptor isoform is available. In the present study, we cloned human long-GPR17, determined its tissue distribution, and characterized its pharmacological specificity in 1321N1 cells by [35S]GTPgammaS binding (which measures the ability of G protein-coupled receptor agonists to increase GTP binding to G proteins) and whole cell patch-clamp recording measuring receptor coupling to K+ channels. [35S]GTPgammaS binding in long-GPR17-expressing 1321N1 cells revealed concentration-dependent responses to uracil nucleotides (UDP-galactose = UDP > UDP-glucose) and cysteinyl leukotrienes (LTC4 > LTD4), which were counteracted by a purinergic (cangrelor) and a cysteinyl leukotriene antagonist (montelukast), respectively. The nonhydrolyzable ATP analog ATPgammaS also acted as an antagonist. GPR17 coupled to Gi and, to a lesser extent, Gq proteins. UDP-glucose and LTD(4) also induced increases in overall outward K+ currents, which were antagonized by the purinergic antagonists MRS2179 and cangrelor and by montelukast. We conclude that the previously uncharacterized long-GPR17 isoform is a functional receptor that is stimulated by both uracil nucleotides and cysteinyl leukotrienes. We also show that the signaling pathway of GPR17 involves the generation of outward K+ currents, an important protective mechanism that, in brain, is specifically aimed at reducing neuronal hyperexcitability and resultant neuronal injury.
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http://dx.doi.org/10.1152/ajpcell.00658.2008DOI Listing
October 2009