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      Specific Temporal Distribution and Subcellular Localization of a Functional Vesicular Nucleotide Transporter (VNUT) in Cerebellar Granule Neurons

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          Abstract

          Adenosine triphosphate (ATP) is an important extracellular neurotransmitter that participates in several critical processes like cell differentiation, neuroprotection or axon guidance. Prior to its exocytosis, ATP must be stored in secretory vesicles, a process that is mediated by the Vesicular Nucleotide Transporter (VNUT). This transporter has been identified as the product of the SLC17A9 gene and it is prominently expressed in discrete brain areas, including the cerebellum. The main population of cerebellar neurons, the glutamatergic granule neurons, depends on purinergic signaling to trigger neuroprotective responses. However, while nucleotide receptors like P2X7 and P2Y13 are known to be involved in neuroprotection, the mechanisms that regulate ATP release in relation to such events are less clearly understood. In this work, we demonstrate that cerebellar granule cells express a functional VNUT that is involved in the regulation of ATP exocytosis. Numerous vesicles loaded with this nucleotide can be detected in these granule cells and are staining by the fluorescent ATP-marker, quinacrine. High potassium stimulation reduces quinacrine fluorescence in granule cells, indicating they release ATP via calcium dependent exocytosis. Specific subcellular markers were used to assess the localization of VNUT in granule cells, and the transporter was detected in both the axonal and somatodendritic compartments, most predominantly in the latter. However, co-localization with the specific lysosomal marker LAMP-1 indicated that VNUT can also be found in non-synaptic vesicles, such as lysosomes. Interestingly, the weak co-localization between VNUT and VGLUT1 suggests that the ATP and glutamate vesicle pools are segregated, as also observed in the cerebellar cortex. During post-natal cerebellar development, VNUT is found in granule cell precursors, co-localizing with markers of immature cells like doublecortin, suggesting that this transporter may be implicated in the initial stages of granule cell development.

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          P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury.

          Pain after nerve damage is an expression of pathological operation of the nervous system, one hallmark of which is tactile allodynia-pain hypersensitivity evoked by innocuous stimuli. Effective therapy for this pain is lacking, and the underlying mechanisms are poorly understood. Here we report that pharmacological blockade of spinal P2X4 receptors (P2X4Rs), a subtype of ionotropic ATP receptor, reversed tactile allodynia caused by peripheral nerve injury without affecting acute pain behaviours in naive animals. After nerve injury, P2X4R expression increased strikingly in the ipsilateral spinal cord, and P2X4Rs were induced in hyperactive microglia but not in neurons or astrocytes. Intraspinal administration of P2X4R antisense oligodeoxynucleotide decreased the induction of P2X4Rs and suppressed tactile allodynia after nerve injury. Conversely, intraspinal administration of microglia in which P2X4Rs had been induced and stimulated, produced tactile allodynia in naive rats. Taken together, our results demonstrate that activation of P2X4Rs in hyperactive microglia is necessary for tactile allodynia after nerve injury and is sufficient to produce tactile allodynia in normal animals. Thus, blocking P2X4Rs in microglia might be a new therapeutic strategy for pain induced by nerve injury.
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            Secretory lysosomes.

            Regulated secretion of stored secretory products is important in many cell types. In contrast to professional secretory cells, which store their secretory products in specialized secretory granules, some secretory cells store their secretory proteins in a dual-function organelle, called a secretory lysosome. Functionally, secretory lysosomes are unusual in that they serve both as a degradative and as a secretory compartment. Recent work shows that cells with secretory lysosomes use new sorting and secretory pathways. The importance of these organelles is highlighted by several genetic diseases, in which immune function and pigmentation--two processes that normally involve secretory lysosomes--are impaired.
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              P2X4R+ microglia drive neuropathic pain.

              Neuropathic pain, the most debilitating of all clinical pain syndromes, may be a consequence of trauma, infection or pathology from diseases that affect peripheral nerves. Here we provide a framework for understanding the spinal mechanisms of neuropathic pain as distinct from those of acute pain or inflammatory pain. Recent work suggests that a specific microglia response phenotype characterized by de novo expression of the purinergic receptor P2X4 is critical for the pathogenesis of pain hypersensitivity caused by injury to peripheral nerves. Stimulating P2X4 receptors initiates a core pain signaling pathway mediated by release of brain-derived neurotrophic factor, which produces a disinhibitory increase in intracellular chloride in nociceptive (pain-transmitting) neurons in the spinal dorsal horn. The changes caused by signaling from P2X4R(+) microglia to nociceptive transmission neurons may account for the main symptoms of neuropathic pain in humans, and they point to specific interventions to alleviate this debilitating condition.
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                Author and article information

                Contributors
                Journal
                Front Pharmacol
                Front Pharmacol
                Front. Pharmacol.
                Frontiers in Pharmacology
                Frontiers Media S.A.
                1663-9812
                22 December 2017
                2017
                : 8
                : 951
                Affiliations
                [1] 1Department of Biochemistry and Molecular Biology, Faculty of Veterinary, Complutense University of Madrid , Madrid, Spain
                [2] 2University Institute of Neurochemistry Research (IUIN), Complutense University of Madrid , Madrid, Spain
                [3] 3Instituto de Investigación Sanitaria del Hospital Clínico San Carlos , Madrid, Spain
                Author notes

                Edited by: Francisco Ciruela, University of Barcelona, Spain

                Reviewed by: Marçal Pastor-Anglada, University of Barcelona, Spain; Arun Chaudhury, GIM Foundation, United States

                *Correspondence: Rosa Gómez-Villafuertes, marosa@ 123456ucm.es ; marosa@ 123456vet.ucm.es María T. Miras-Portugal, mtmiras@ 123456ucm.es ; mtmiras@ 123456vet.ucm.es

                This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology

                Article
                10.3389/fphar.2017.00951
                5744399
                77976310-a256-44a1-8b97-a55464ce9171
                Copyright © 2017 Menéndez-Méndez, Díaz-Hernández, Ortega, Gualix, Gómez-Villafuertes and Miras-Portugal.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 25 October 2017
                : 15 December 2017
                Page count
                Figures: 7, Tables: 0, Equations: 0, References: 87, Pages: 14, Words: 0
                Funding
                Funded by: Ministerio de Economía y Competitividad 10.13039/501100003329
                Award ID: BFU 2014-53654-P
                Award ID: RYC-2013-13290
                Award ID: BFU2015-70067REDC
                Funded by: Fundación Ramón Areces 10.13039/100008054
                Award ID: PR2018/16-02
                Funded by: Consejería de Sanidad, Comunidad de Madrid 10.13039/501100006541
                Award ID: S2013/ICE-2958
                Categories
                Pharmacology
                Original Research

                Pharmacology & Pharmaceutical medicine
                vnut,granule cells,cerebellum,atp exocytosis,vglut1,gcps
                Pharmacology & Pharmaceutical medicine
                vnut, granule cells, cerebellum, atp exocytosis, vglut1, gcps

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