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Nucleotide receptors in trigeminal satellite glial cells as new targets for the pharmacological control of migraine pain: in vitro and in vivo studies
No full textBackground and Purpose - The main aim of my PhD research project is to study pain transduction mechanisms in trigeminal ganglia (TG) in migraine, to understand why sensory trigeminal neurons become hyperactive in this pathological status, and how their activity can be pharmacologically modulated. We focused our studies on the role of the purinergic system in the neuron-to-glial cells communication within the TG, and its cross-talk with known pro-algogenic systems (such as bradykinin, BK, calcitonin gene-related peptide, CGRP, and prostaglandins). The final goal is the identification of new cellular and molecular players in the onset and maintenance of trigeminal-associated pain, for the development of new effective therapeutic strategies for migraine.
Methods and Results – For the in vitro studies, we set up primary mixed neuron-glia or purified satellite glial cell (SGCs) cultures from theTG of C57BL6 mice. G protein-coupled P2Y receptor function was evaluated by single cell calcium imaging, and the extracellular concentrations of CGRP and PGE2 were measured by ELISA assays. Western blot experiments on P2Y1 and P2Y2 receptor subtypes were also performed. Concerning the role of metabotropic purinergic receptors, our data show that exposure of mixed-neuron glia cultures to BK induced the neuronal release of CGRP, which in turn significantly potentiated the ADP-responsive P2Y1 and the UTP-sensitive P2Y2 receptor subtypes on surrounding SGCs. The increased activity of P2 receptors was related to increased receptor protein expression. Interestingly, the anti-migraine drug sumatriptan fully inhibited both CGRP release and glial P2Y-receptor potentiation. Moreover, exposure to BK led to increased production of PGE2, an effect completely abolished by the COX-1 inhibitor acetylsalicylic acid (ASA), whichalso blocked neuronal CGRP release. Taken together, these results suggest that a complex cross-talk between neuronal and glial cells takes place in the TG, involving pain mediators and extracellular nucleotides. Modulation of this network by known anti-migraine drugs, such as triptans and COX inhibitors, suggests that it might play an important role in the development of migraine pain.
In vivo studies were aimed at evaluating the pro- or anti-algogenic role of P2Y receptors through their selective inhibition. To this purpose, we set up a sub-chronic model of inflammatory trigeminal hypersensitivity, by injecting complete Freund adjuvant (CFA) into the temporomandibular joint (TMJ) of rats. CFA-injected animals showed ipsilateral mechanical allodynia and TMJ edema. Glial cell activation was evaluated in the spinal-trigeminal system by immunohistochemistry and western blotting analysis of GFAP protein expression, a typical marker of activated SGCs. A significant glial activation within the TG was observed starting from 24 hours up to 11 days after CFA injection, thus confirming that our model leads to TG sensitization. Moreover, upregulation of P2Y1 and P2Y2 protein expression was also observed, thus corroborating our in vitro data. Interestingly, the non-selective P2Y antagonist PPADS showed a strong analgesic effect on CFA-induced TG inflammation, which is comparable to ASA-mediated analgesia.
Conclusions - These results suggest a possible the pro-algogenic role for P2Y receptors in the development of trigeminal sensitization and migraine pain, opening the future perspective of identifying innovative and more selective pharmacological approaches for the sake of those migraineurs who are insensitive to currently available drugs.
References
[1] G. Magni, S. Ceruti. P2Y purinergic receptors: new targets for analgesic and antimigraine drugs. Biochem Pharmacol, 85(4):466-77, 2013.
[2] S. Ceruti, G. Villa, M. Fumagalli, L. Colombo, G. Magni, M. Zanardelli, E. Fabbretti, C. Verderio, A. M. J. M. van den Maagdenberg, A. Nistri, M. P. Abbracchio. CGRP−mediated enhancement of purinergic neuron/glia communication by the algogenic factor bradykinin in mouse trigeminal ganglia from wild type and R192Q Cav2.1 knock-in mice: implications for basic mechanisms of migraine pain. J Neuroscience, 31(10):3638 –3649, 2011
[3] G. Villa, S. Ceruti, M. Zanardelli, G. Magni, L. Jasmin, P. T. Ohara, M. P. Abbracchio.. Temporomandibular join inflammation activates glial and immune cells in both the trigeminal ganglia and the spinal trigeminal nucleus. Mol Pain, 6:89, 2010
P2Y purinergic receptors in neuron-to-glia communication in trigeminal ganglia and their role in migraine pain
No full textTrigeminal satellite glial cells (SGCs) express G protein-coupled P2Y receptors for extracellular nucleotides (ATP, ADP, and UTP), whose activity is increased by exposure to bradykinin (BK) in vitro. However, their possible role in the development and maintenance of migraine pain is still unknown. Here we show the existence of a cross-communication between neurons and SGCs leading to glial P2Y receptor upregulation. In fact, BK induced the neuronal release of CGRP, which in turn activated an ERK1/2-dependent pathway in surrounding SGCs and potentiated P2Y receptors. Indeed, BK-mediated effects were inhibited by the anti-migraine drug sumatriptan, which targets neurons. Interestingly, both basal and BK-stimulated CGRP release was higher in trigeminal cultures from CaV2.1 α1 R192Q mutant knock-in (KI) mice, a model of familial hemiplegic migraine type 1. Indeed, BK significantly up-regulated the number of SGCs with functional P2Y receptors in cultures from KI mice only, suggesting that this cross-talk might become even more important in migraine-prone conditions, and that P2Y receptors might represent innovative targets for the development of therapeutic agents against migraine pain
Nucleotide receptors in trigeminal satellite glial cells: new targets for the pharmacological control of migraine pain
No full textAccording to the WHO, migraine is a rather common disorder affecting 15% of adults in the Western World. It has been included in the list of the 20 more disabling pathologies, due to the dramatic reduction of the patient’s quality of life, and also to massive personal and social costs, in terms of both medical expenses and lost workdays. Despite the recent introduction to the market of new, potent and effective anti-migraine drugs (e.g., triptans), still a significant number of migraineurs is insensitive to the currently available pharmacological approaches, suggesting that there are yet-to-be identified molecular and cellular players at the basis of the disease, which are not targeted by currently utilized drugs. For example, it is now believed that the trigeminal (TG)-brainstem sensory system plays a central role in the development and maintenance of migraine pain. Currently available pharmacological approaches to migraine mostly modulate neuronal activity, but it is now clear that TG neurons act in strict synergy with non-neuronal cells, in particular “satellite” glial cells (SGCs) that envelop neuronal bodies within the ganglion, to promote and maintain migraine-associated pain. Therefore, others and we have become interested in verifying whether SGCs might represent innovative pharmacological targets to migraine pain, and we have focused our research on the purinergic system, based on the knowledge that the crosstalk between neurons and SGCs is at least partly mediated by the activation of purinergic receptors responding to endogenously released nucleotides, like ATP and UTP. We are also interested in studying the cross-talk between the purinergic system and other known pro-algogenic signals, in order to test whether the modulation of purinergic receptors is involved in the mechanism of action of known pain-killers currently utilized by patients.
To test our hypotheses, we utilized both mixed neuron-glia and purified glial cultures form mouse TG. We previously showed that the algogenic mediator bradykinin (BK) potentiates G protein-coupled purinergic P2Y-receptors on SGCs in primary trigeminal cultures (Ceruti et al., Cell Calcium 43:576-90, 2008), through the neuronal release of the pro-algogenic mediator calcitonin gene-related peptide (CGRP; Ceruti et al., J Neurosci 31:3638-49, 2011). Interestingly, the anti-migraine drug sumatriptan fully inhibited both CGRP release and glial P2Y-receptor potentiation, therefore suggesting a possible role for receptors activated by adenine and uracil nucleotides in the mechanism of action of this drug. Indeed, exposure to BK led to an increased production of PGE2, which was fully inhibited by the predominant COX-1 inhibitor acetyl salicylic acid (ASA), sometimes utilized to abort migraine attacks. The latter also blocked neuronal CGRP release, thus highlighting the existence of a complex cross-talk between arachidonic acid metabolites, the CGRP system and purinergic receptors, which modulate glial and neuronal cell functions in the TG. By a pharmacological approach employing selective antagonists, we have identified the P2Y receptor subtypes that are up-regulated in TG glial cells by BK and CGRP exposure, namely the ADP-responsive P2Y1 and the UTP-sensitive P2Y2 subtypes. Our data also suggest that their increased activity is both due to an increased receptor protein expression, but also, mostly for the P2Y1 subtype, to the modulation of their subcellular localization to membrane lipid rafts. Studies on the possible pro- or anti-algogenic role of these receptor subtypes are currently in progress. Interestingly, this complex molecular cross-talk between the purinergic system and known pro-algogenic substances was further potentiated in cell cultures from CaV2.1 a1 R192Q mutant knock-in (KI) mice expressing a human mutation causing familial hemiplegic migraine type 1, further suggesting the possible involvement of glial purinergic receptors in the onset and maintenance of migraine-associated pain. Overall, our findings suggest, for the first time, that P2Y receptors on glial cells act might represent new targets for the development of innovative therapeutic agents against migraine pain.
The financial contribution of the Italian Comitato Telethon (project #GGP10082A) and of the Fondazione Cariplo (project #2011-0505) is gratefully acknowledged
The Purinergic System and Glial Cells : Emerging Costars in Nociception
Full text linkIt is now well established that glial cells not only provide mechanical and trophic support to neurons but can directly contribute to neurotransmission, for example, by release and uptake of neurotransmitters and by secreting pro- and anti-inflammatory mediators. This has greatly changed our attitude towards acute and chronic disorders, paving the way for new therapeutic approaches targeting activated glial cells to indirectly modulate and/or restore neuronal functions. A deeper understanding of the molecular mechanisms and signaling pathways involved in neuron-to-glia and glia-to-glia communication that can be pharmacologically targeted is therefore a mandatory step toward the success of this new healing strategy. This holds true also in the field of pain transmission, where the key involvement of astrocytes and microglia in the central nervous system and satellite glial cells in peripheral ganglia has been clearly demonstrated, and literally hundreds of signaling molecules have been identified. Here, we shall focus on one emerging signaling system involved in the cross talk between neurons and glial cells, the purinergic system, consisting of extracellular nucleotides and nucleosides and their membrane receptors. Specifically, we shall summarize existing evidence of novel "druggable" glial purinergic targets, which could help in the development of innovative analgesic approaches to chronic pain states
Uracil nucleotides : from metabolic intermediates to neuroprotection and neuroinflammation
No full textUracil nucleotides (i.e., UTP and UDP) have been known for years as fundamental intermediates in the de novo synthesis of the other pyrimidine nucleotides, which altogether represent key building blocks for nucleic acid synthesis. In addition, their sugar conjugates (i.e., UDP-glucose and UDP-galactose) enter in several biochemical routes, for example leading to glycogen biosynthesis, and protein and lipid glycosylation, which in turn contribute to the synthesis of essential components of the cellular plasma membrane. More recently, the existence of a "pyrimidinergic transmission" has arisen from the discovery that several purinergic G protein-coupled P2Y receptors can be activated also or exclusively by uracil nucleotides and sugar conjugates. The number of these receptors is continuously growing over years with the discovery that previously "orphan" G protein-coupled receptors are actually responding to this class of molecules. Therefore, new unforeseen effects mediated by uracil derivatives have emerged, in particular in the nervous system, and previously unexplored avenues for the pharmacological manipulation of this system are currently under investigation. In this commentary we shall try to put together our current knowledge on the biochemical and receptor-mediated effects of uracil nucleotide derivatives with a specific focus on the nervous system in order to depict a clearer view of the importance of the pyrimidinergic system in both physiological and pathological conditions
Adenosine signaling in glioma cells
No full textPurines and pyrimidines are fundamental signaling molecules in controlling the survival and proliferation of astrocytes, as well as in mediating cell-to-cell communication between glial cells and neurons in the healthy brain. The malignant transformation of astrocytes towards progressively more aggressive brain tumors (from astrocytoma to anaplastic glioblastoma) leads to modifications in both the survival and cell death pathways which overall confer a growth advantage to malignant cells and resistance to many cytotoxic stimuli. It has been demonstrated, however, that, in astrocytomas, several purinergic (in particular adenosinergic) pathways controlling cell survival and death are still effective and, in some cases, even enhanced, providing invaluable targets for purine-based chemotherapy, that still represents an appropriate pharmacological approach to brain tumors. In this chapter, the current knowledge on both receptor-mediated and receptor-independent adenosine pathways in astrocytomas will be reviewed, with a particular emphasis on the most promising targets which could be translated from in vitro studies to in vivo pharmacology
Targeting NGF system to fight neuropathic pain behavioral and immunohistochemical evidence in mice
Full text linkBackground and Aims: it has been demonstrated that the anti-NGF αD11 and the anti-TrkA MNAC13 counteract neuropathic pain in mice. The aim of this study was to evaluate the duration of the action of the two antibodies and the structural and morphological alterations induced in central and peripheral nervous system.
Methods: Chronic Constriction Injury (CCI) of the sciatic nerve was performed in C57BL/6J mice. Mice were administered with αD11 or MNAC13 (70 or 100 μg/mouse/day) from day 3 until day 10 post-CCI. Analgesic effects were tested through Dynamic Plantar Aesthesiometer from day 3 to day 90. Spinal cords and sciatic nerves were collected at D3, D11, D24 and D90 for immunohistochemistry.
Results: αD11 and MNAC13 induce significant dose- and time-dependent analgesic effects: the antiallodynic effect was still present at D90 following the highest doses of both antibodies. Immunohistochemical analysis show significant differences in inflammatory and myelination markers between treated and control animals, treated animals showing reduced glial and mast cells activation and a better nerve regeneration.
Conclusions: Data obtained prove that: i) the analgesic effect of the antibodies αD11 and MNAC13 are extremely long-lasting, being observable more than two months after the end of the treatment and ii) the antiNGF and antiTrkA antibodies reduce inflammation and facilitate the regenerative processes. Therefore, our results strongly support the importance of considering the NGF system in the development of novel therapies to modulate and control neuropathy
STUDY OF THE ISOSPIN SYMMETRY IN 80ZR
No full textIn Nature, symmetries help us to describe a complex physical system in a simple way and to understand better its behavior. Indeed, symmetries are strongly related to conservation laws which, in quantum mechanics, translate into good quantum number to describe the system. At the same time, the possible breaking of a symmetry opens the gates for new and unexpected scenarios.
In nuclear physics many symmetries were identified. One of these is the isospin symmetry, which plays a key role in nuclear structure and nuclear reaction. The isospin symmetry was introduced by Heisenberg in 1932 to describe the experimental evidence of the charge independence of the nuclear interaction: neutrons and protons are considered as two different states of the same particle, the nucleon.
In the atomic nuclei, the presence of the Coulomb interaction between protons breaks this symmetry and induces a mixing between states with different isospin. In this situation it is impossible to assign to a nuclear state a unique value of isospin. This phenomenon is called isospin mixing.
The knowledge of the isospin mixing is a fundamental quantity needed both to explain the properties of the Isobaric Analogue State and for its connection with the test of the unitarity of the Cabibbo-Kobayashi-Maskawa matrix.
In this Thesis, we report a new study addressing the problem of the isospin mixing in the nucleus 80Zr, using the Giant Dipole Resonance -decay
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