Publications by authors named "Marta Fumagalli"

48 Publications

Oligodendrocyte Dysfunction in Amyotrophic Lateral Sclerosis: Mechanisms and Therapeutic Perspectives.

Cells 2021 Mar 5;10(3). Epub 2021 Mar 5.

Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20133 Milan, Italy.

Myelin is the lipid-rich structure formed by oligodendrocytes (OLs) that wraps the axons in multilayered sheaths, assuring protection, efficient saltatory signal conduction and metabolic support to neurons. In the last few years, the impact of OL dysfunction and myelin damage has progressively received more attention and is now considered to be a major contributing factor to neurodegeneration in several neurological diseases, including amyotrophic lateral sclerosis (ALS). Upon OL injury, oligodendrocyte precursor cells (OPCs) of adult nervous tissue sustain the generation of new OLs for myelin reconstitution, but this spontaneous regeneration process fails to successfully counteract myelin damage. Of note, the functions of OPCs exceed the formation and repair of myelin, and also involve the trophic support to axons and the capability to exert an immunomodulatory role, which are particularly relevant in the context of neurodegeneration. In this review, we deeply analyze the impact of dysfunctional OLs in ALS pathogenesis. The possible mechanisms underlying OL degeneration, defective OPC maturation, and impairment in energy supply to motor neurons (MNs) have also been examined to provide insights on future therapeutic interventions. On this basis, we discuss the potential therapeutic utility in ALS of several molecules, based on their remyelinating potential or capability to enhance energy metabolism.
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http://dx.doi.org/10.3390/cells10030565DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8000560PMC
March 2021

Purinergic Receptors on Oligodendrocyte Progenitors: Promising Targets for Myelin Repair in Multiple Sclerosis?

Front Pharmacol 2020 27;11:629618. Epub 2021 Jan 27.

Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy.

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http://dx.doi.org/10.3389/fphar.2020.629618DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872961PMC
January 2021

The immune-inflammatory response of oligodendrocytes in a murine model of preterm white matter injury: the role of TLR3 activation.

Cell Death Dis 2021 02 8;12(2):166. Epub 2021 Feb 8.

Université de Paris, Inserm UMR 1141 NeuroDiderot, F-75019, Paris, France.

A leading cause of preterm birth is the exposure to systemic inflammation (maternal/fetal infection), which leads to neuroinflammation and white matter injury (WMI). A wide range of cytokines and chemokines are expressed and upregulated in oligodendrocytes (OLs) in response to inflammation and numerous reports show that OLs express several receptors for immune related molecules, which enable them to sense inflammation and to react. However, the role of OL immune response in WMI is unclear. Here, we focus our study on toll-like receptor-3 (TLR3) that is activated by double-strand RNA (dsRNA) and promotes neuroinflammation. Despite its importance, its expression and role in OLs remain unclear. We used an in vivo mouse model, which mimics inflammation-mediated WMI of preterm born infants consisting of intraperitoneal injection of IL-1β from P1 to P5. In the IL-1β-treated animals, we observed the upregulation of Tlr3, IL-1β, IFN-β, Ccl2, and Cxcl10 in both PDGFRα+ and O4+ sorted cells. This upregulation was higher in O4+ immature OLs (immOLs) as compared to PDGFRα+ OL precursor cells (OPCs), suggesting a different sensitivity to neuroinflammation. These observations were confirmed in OL primary cultures: cells treated with TLR3 agonist Poly(I:C) during differentiation showed a stronger upregulation of Ccl2 and Cxcl10 compared to cells treated during proliferation and led to decreased expression of myelin genes. Finally, OLs were able to modulate microglia phenotype and function depending on their maturation state as assessed by qPCR using validated markers for immunomodulatory, proinflammatory, and anti-inflammatory phenotypes and by phagocytosis and morphological analysis. These results show that during inflammation the response of OLs can play an autonomous role in blocking their own differentiation: in addition, the immune activation of OLs may play an important role in shaping the response of microglia during inflammation.
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http://dx.doi.org/10.1038/s41419-021-03446-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870670PMC
February 2021

Microglial vesicles improve post-stroke recovery by preventing immune cell senescence and favoring oligodendrogenesis.

Mol Ther 2021 04 10;29(4):1439-1458. Epub 2020 Dec 10.

Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20133 Milan, Italy. Electronic address:

Contrasting myelin damage through the generation of new myelinating oligodendrocytes represents a promising approach to promote functional recovery after stroke. Here, we asked whether activation of microglia and monocyte-derived macrophages affects the regenerative process sustained by G protein-coupled receptor 17 (GPR17)-expressing oligodendrocyte precursor cells (OPCs), a subpopulation of OPCs specifically reacting to ischemic injury. GPR17-iCreER:CAG-eGFP reporter mice were employed to trace the fate of GPR17-expressing OPCs, labeled by the green fluorescent protein (GFP), after permanent middle cerebral artery occlusion. By microglia/macrophages pharmacological depletion studies, we show that innate immune cells favor GFP OPC reaction and limit myelin damage early after injury, whereas they lose their pro-resolving capacity and acquire a dystrophic "senescent-like" phenotype at later stages. Intracerebral infusion of regenerative microglia-derived extracellular vesicles (EVs) restores protective microglia/macrophages functions, limiting their senescence during the post-stroke phase, and enhances the maturation of GFP OPCs at lesion borders, resulting in ameliorated neurological functionality. In vitro experiments show that EV-carried transmembrane tumor necrosis factor (tmTNF) mediates the pro-differentiating effects on OPCs, with future implications for regenerative therapies.
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http://dx.doi.org/10.1016/j.ymthe.2020.12.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058432PMC
April 2021

TNF Production and Release from Microglia via Extracellular Vesicles: Impact on Brain Functions.

Cells 2020 09 23;9(10). Epub 2020 Sep 23.

Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20133 Milan, Italy.

Tumor necrosis factor (TNF) is a pleiotropic cytokine powerfully influencing diverse processes of the central nervous system (CNS) under both physiological and pathological conditions. Here, we analyze current literature describing the molecular processes involved in TNF synthesis and release from microglia, the resident immune cells of the CNS and the main source of this cytokine both in brain development and neurodegenerative diseases. A special attention has been given to the unconventional vesicular pathway of TNF, based on the emerging role of microglia-derived extracellular vesicles (EVs) in the propagation of inflammatory signals and in mediating cell-to-cell communication. Moreover, we describe the contribution of microglial TNF in regulating important CNS functions, including the neuroinflammatory response following brain injury, the neuronal circuit formation and synaptic plasticity, and the processes of myelin damage and repair. Specifically, the available data on the functions mediated by microglial EVs carrying TNF have been scrutinized to gain insights on possible novel therapeutic strategies targeting TNF to foster CNS repair.
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http://dx.doi.org/10.3390/cells9102145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598215PMC
September 2020

Development of the first in vivo GPR17 ligand through an iterative drug discovery pipeline: A novel disease-modifying strategy for multiple sclerosis.

PLoS One 2020 22;15(4):e0231483. Epub 2020 Apr 22.

Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan, Italy.

The GPR17 receptor, expressed on oligodendroglial precursors (OPCs, the myelin producing cells), has emerged as an attractive target for a pro-myelinating strategy in multiple sclerosis (MS). However, the proof-of-concept that selective GPR17 ligands actually exert protective activity in vivo is still missing. Here, we exploited an iterative drug discovery pipeline to prioritize novel and selective GPR17 pro-myelinating agents out of more than 1,000,000 compounds. We first performed an in silico high-throughput screening on GPR17 structural model to identify three chemically-diverse ligand families that were then combinatorially exploded and refined. Top-scoring compounds were sequentially tested on reference pharmacological in vitro assays with increasing complexity, ending with myelinating OPC-neuron co-cultures. Successful ligands were filtered through in silico simulations of metabolism and pharmacokinetics, to select the most promising hits, whose dose and ability to target the central nervous system were then determined in vivo. Finally, we show that, when administered according to a preventive protocol, one of them (named by us as galinex) is able to significantly delay the onset of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. This outcome validates the predictivity of our pipeline to identify novel MS-modifying agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231483PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176092PMC
July 2020

Under-Age Children Returning From Jihadist Group Operation Areas: How Can We Make a Diagnosis and Construct a Narrative With a Fragmentary Anamnesis?

Front Psychiatry 2020 26;11:149. Epub 2020 Mar 26.

Child and Adolescent Psychiatry Department, AP-HP, Avicenne Hospital, EA 4403 Paris 13 University, Bobigny, France.

Introduction: Since 2011, the French government estimates that about 500 French children have been born in or taken by their parents to areas where terrorist operations prevail. Since May 2017, 75 children who returned to France have benefited from a dedicated health care system.

Method: This article is the result of clinical interviews conducted with 53 patients evaluated and taken care of at Avicenne Hospital in Bobigny. To our knowledge, no studies have been published on this subject.

Results: A total of 32 evaluations have been completed, all of which indicated the need for care for these children. Of these children, 64% are under 5 years old, and 59% were born in France. Their clinical profiles are heterogeneous and fluctuate with time.

Discussion: The multiple adverse events experienced by these children and the uniqueness of children born to families suspected by authorities of having participated in activities related to terrorism make this situation unprecedented. How can we make a diagnosis of PTSD without the help of a precise anamnesis? How can we help these children form a structuring narrative that avoids the pitfalls inherent to generalized fascination?
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http://dx.doi.org/10.3389/fpsyt.2020.00149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117100PMC
March 2020

Abnormal Upregulation of GPR17 Receptor Contributes to Oligodendrocyte Dysfunction in SOD1 G93A Mice.

Int J Mol Sci 2020 Mar 31;21(7). Epub 2020 Mar 31.

Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, 20133 Milan, Italy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive loss of motor neurons (MN). Importantly, MN degeneration is intimately linked to oligodendrocyte dysfunction and impaired capacity of oligodendrocyte precursor cells (OPCs) to regenerate the myelin sheath enwrapping and protecting neuronal axons. Thus, improving OPC reparative abilities represents an innovative approach to counteract MN loss. A pivotal regulator of OPC maturation is the P2Y-like G protein-coupled receptor 17 (GPR17), whose role in ALS has never been investigated. In other models of neurodegeneration, an abnormal increase of GPR17 has been invariably associated to myelin defects and its pharmacological manipulation succeeded in restoring endogenous remyelination. Here, we analyzed GPR17 alterations in the SOD1 ALS mouse model and assessed in vitro whether this receptor could be targeted to correct oligodendrocyte alterations. Western-blot and immunohistochemical analyses showed that GPR17 protein levels are significantly increased in spinal cord of ALS mice at pre-symptomatic stage; this alteration is exacerbated at late symptomatic phases. Concomitantly, mature oligodendrocytes degenerate and are not successfully replaced. Moreover, OPCs isolated from spinal cord of SOD1 mice display defective differentiation compared to control cells, which is rescued by treatment with the GPR17 antagonist montelukast. These data open novel therapeutic perspectives for ALS management.
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http://dx.doi.org/10.3390/ijms21072395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177925PMC
March 2020

Regulation and signaling of the GPR17 receptor in oligodendroglial cells.

Glia 2020 10 22;68(10):1957-1967. Epub 2020 Feb 22.

Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy.

Remyelination, namely, the formation of new myelin sheaths around denuded axons, counteracts axonal degeneration and restores neuronal function. Considerable advances have been made in understanding this regenerative process that often fails in diseases like multiple sclerosis, leaving axons demyelinated and vulnerable to damage, thus contributing to disease progression. The identification of the membrane receptor GPR17 on a subset of oligodendrocyte precursor cells (OPCs), which mediate remyelination in the adult central nervous system (CNS), has led to a huge amount of evidence that validated this receptor as a new attractive target for remyelinating therapies. Here, we summarize the role of GPR17 in OPC function, myelination and remyelination, describing its atypical pharmacology, its downstream signaling, and the genetic and epigenetic factors modulating its activity. We also highlight crucial insights into GPR17 pathophysiology coming from the demonstration that oligodendrocyte injury, associated with inflammation in chronic neurodegenerative conditions, is invariably characterized by abnormal and persistent GPR17 upregulation, which, in turn, is accompanied by a block of OPCs at immature premyelinating stages. Finally, we discuss the current literature in light of the potential exploitment of GPR17 as a therapeutic target to promote remyelination.
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http://dx.doi.org/10.1002/glia.23807DOI Listing
October 2020

Regulation of Oligodendrocyte Functions: Targeting Lipid Metabolism and Extracellular Matrix for Myelin Repair.

J Clin Med 2020 Feb 8;9(2). Epub 2020 Feb 8.

Department of Phamacological and Biomolecular Sciences, Università degli Studi di Milano, Via Balzaretti, 9-20133 Milan, Italy.

Myelin is an essential structure that protects axons, provides metabolic support to neurons and allows fast nerve transmission. Several neurological diseases, such as multiple sclerosis, are characterized by myelin damage, which is responsible of severe functional impairment. Myelin repair requires the timely recruitment of adult oligodendrocyte precursor cells (OPCs) at the lesion sites, their differentiation and maturation into myelinating oligodendrocytes. As a consequence, OPCs undergo profound changes in their morphology, functions, and interactions with other cells and extracellular environment, thus requiring the reorganization of both their lipid metabolism and their membrane composition, which is substantially different compared to other plasma membranes. Despite the growing knowledge in oligodendroglia biology and in the mechanisms involved in OPC-mediated regeneration, the identification of strategies to promote remyelination still remains a challenge. Here, we describe how altered lipid metabolism in oligodendrocytes influences the pathogenesis of demyelination, and we show that several FDA-approved drugs with a previously unknown remyelination potential do act on cholesterol and lipid biosynthetic pathways. Since the interplay between myelin lipids and axons is strictly coordinated by the extracellular matrix (ECM), we also discuss the role of different ECM components, and report the last findings on new ECM-modifiers able to foster endogenous remyelination.
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http://dx.doi.org/10.3390/jcm9020470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7073561PMC
February 2020

Surface Plasmon Resonance as a Tool for Ligand Binding Investigation of Engineered GPR17 Receptor, a G Protein Coupled Receptor Involved in Myelination.

Front Chem 2019 10;7:910. Epub 2020 Jan 10.

Department of Biotechnology, University of Verona, Verona, Italy.

The aim of this study was to investigate the potential of surface plasmon resonance (SPR) spectroscopy for the measurement of real-time ligand-binding affinities and kinetic parameters for GPR17, a G protein-coupled receptor (GPCR) of major interest in medicinal chemistry as potential target in demyelinating diseases. The receptor was directly captured, in a single-step, from solubilized membrane extracts on the sensor chip through a covalently bound anti-6x-His-antibody and retained its ligand binding activity for over 24 h. Furthermore, our experimental setup made possible, after a mild regeneration step, to remove the bound receptor without damaging the antibody, and thus to reuse many times the same chip. Two engineered variants of GPR17, designed for crystallographic studies, were expressed in insect cells, extracted from crude membranes and analyzed for their binding with two high affinity ligands: the antagonist Cangrelor and the agonist Asinex 1. The calculated kinetic parameters and binding constants of ligands were in good agreement with those reported from activity assays and highlighted a possible functional role of the N-terminal residues of the receptor in ligand recognition and binding. Validation of SPR results was obtained by docking and molecular dynamics of GPR17-ligands interactions and by functional studies. The latter allowed us to confirm that Asinex 1 behaves as GPR17 receptor agonist, inhibits forskolin-stimulated adenylyl cyclase pathway and promotes oligodendrocyte precursor cell maturation and myelinating ability.
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http://dx.doi.org/10.3389/fchem.2019.00910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966494PMC
January 2020

Detrimental and protective action of microglial extracellular vesicles on myelin lesions: astrocyte involvement in remyelination failure.

Acta Neuropathol 2019 12 30;138(6):987-1012. Epub 2019 Jul 30.

CNR Institute of Neuroscience, via Vanvitelli 32, 20129, Milan, Italy.

Microglia are highly plastic immune cells which exist in a continuum of activation states. By shaping the function of oligodendrocyte precursor cells (OPCs), the brain cells which differentiate to myelin-forming cells, microglia participate in both myelin injury and remyelination during multiple sclerosis. However, the mode(s) of action of microglia in supporting or inhibiting myelin repair is still largely unclear. Here, we analysed the effects of extracellular vesicles (EVs) produced in vitro by either pro-inflammatory or pro-regenerative microglia on OPCs at demyelinated lesions caused by lysolecithin injection in the mouse corpus callosum. Immunolabelling for myelin proteins and electron microscopy showed that EVs released by pro-inflammatory microglia blocked remyelination, whereas EVs produced by microglia co-cultured with immunosuppressive mesenchymal stem cells promoted OPC recruitment and myelin repair. The molecular mechanisms responsible for the harmful and beneficial EV actions were dissected in primary OPC cultures. By exposing OPCs, cultured either alone or with astrocytes, to inflammatory EVs, we observed a blockade of OPC maturation only in the presence of astrocytes, implicating these cells in remyelination failure. Biochemical fractionation revealed that astrocytes may be converted into harmful cells by the inflammatory EV cargo, as indicated by immunohistochemical and qPCR analyses, whereas surface lipid components of EVs promote OPC migration and/or differentiation, linking EV lipids to myelin repair. Although the mechanisms through which the lipid species enhance OPC maturation still remain to be fully defined, we provide the first demonstration that vesicular sphingosine 1 phosphate stimulates OPC migration, the first fundamental step in myelin repair. From this study, microglial EVs emerge as multimodal and multitarget signalling mediators able to influence both OPCs and astrocytes around myelin lesions, which may be exploited to develop novel approaches for myelin repair not only in multiple sclerosis, but also in neurological and neuropsychiatric diseases characterized by demyelination.
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http://dx.doi.org/10.1007/s00401-019-02049-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851224PMC
December 2019

MicroRNAs change the games in central nervous system pharmacology.

Biochem Pharmacol 2019 10 25;168:162-172. Epub 2019 Jun 25.

Laboratorio di Farmacologia Molecolare e Cellulare della Trasmissione Purinergica, Dipartimento di Scienze Farmacologiche e Biomolecolari (DiSFeB), Università degli Studi di Milano, Via Balzaretti, 9, 20133 Milano, Italy. Electronic address:

MicroRNAs (miRNAs) represent a class of important post-transcriptional regulators of gene expression, enabling cells to follow their intrinsic developmental program. By directly binding to their targets, miRNAs can both promote transcriptional patterns in crucial steps of cell growth, and act as powerful buffering system that titrate protein content in case of aberrant gene expression. The literature of the last decade showed that the presence of tissue-enriched miRNAs in body fluids could be reminiscent of disease state. This is particularly relevant in neurodegenerative disorders, in which peripheral biomarkers could be helpful means to detect disease onset. However, dysregulation of miRNAs is not merely a consequence of disease, but directly contributes to pathological outcomes. On this basis, increasing interest is growing in the development of pharmacological agents targeting specific miRNAs. Actually, this apparently futuristic approach is already part of the current therapies. In fact, several drugs approved for CNS disorders, such as L-Dopa or valproic acid, were also demonstrated to restore some miRNAs. Moreover, ongoing clinical trials demonstrated that miRNA-based drugs are effective against tumors, suggesting that miRNAs also represent a promising class of therapeutic molecules. However, several issues still need to be addressed, particularly in case of CNS diseases, in which stability and delivery are crucial aspects of the therapy. In this commentary, we highlighted potential advantages and limitations of miRNAs as next generation targets in CNS pharmacology, focusing on multiple sclerosis, a chronic demyelinating disease lacking specific therapeutic targets and bona-fide biomarkers.
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http://dx.doi.org/10.1016/j.bcp.2019.06.019DOI Listing
October 2019

Improvement of fiber connectivity and functional recovery after stroke by montelukast, an available and safe anti-asthmatic drug.

Pharmacol Res 2019 04 25;142:223-236. Epub 2019 Feb 25.

Centro Cardiologico Monzino IRCCS, 20138 Milan, Italy; Department of Pharmacological and Biomolecular Sciences, University of Milan, 20133 Milan, Italy. Electronic address:

Stroke is one of the main causes of death, neurological dysfunctions or disability in elderly. Neuroprotective drugs have been proposed to improve long-term recovery after stroke, but failed to reach clinical effectiveness. Hence, recent studies suggested that restorative therapies should combine neuroprotection and remyelination. Montelukast, an anti-asthmatic drug, was shown to exert neuroprotection in animal models of CNS injuries, but its ability to affect oligodendrocytes, restoring fiber connectivity, remains to be determined. In this study, we evaluated whether montelukast induces long-term repair by promoting fiber connectivity up to 8 weeks after middle cerebral artery occlusion (MCAo), using different experimental approaches such as in vivo diffusion magnetic resonance imaging (MRI), electrophysiological techniques, ex vivo diffusion tensor imaging (DTI)-based fiber tracking and immunohistochemistry. We found that, in parallel with a reduced evolution of ischemic lesion and atrophy, montelukast increased the DTI-derived axial diffusivity and number of myelin fibers, the density of myelin binding protein (MBP) and the number of GSTpi mature oligodendrocytes. Together with the rescue of MCAo-induced impairments of local field potentials in ischemic cortex, the data suggest that montelukast may improve fibers reorganization. Thus, to ascertain whether this effect involved changes of oligodendrocyte precursor cells (OPCs) activation and maturation, we used the reporter GPR17iCreERT2:CAG-eGreen florescent protein (GFP) mice that allowed us to trace the fate of OPCs throughout animal's life. Our results showed that montelukast enhanced the OPC recruitment and proliferation at acute phase, and increased their differentiation to mature oligodendrocytes at chronic phase after MCAo. Considering the crosstalk between OPCs and microglia has been widely reported in the context of demyelinating insults, we also assessed microglia activation. We observed that montelukast influenced the phenotype of microglial cells, increasing the number of M2 polarized microglia/macrophages, over the M1 phenotype, at acute phase after MCAo. In conclusion, we demonstrated that montelukast improves fiber re-organization and long-term functional recovery after brain ischemia, enhancing recruitment and maturation of OPCs. The present data suggest that montelukast, an already approved drug, could be "repositioned "as a protective drug in stroke acting also on fiber re-organization.
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http://dx.doi.org/10.1016/j.phrs.2019.02.025DOI Listing
April 2019

How to reprogram microglia toward beneficial functions.

Glia 2018 12 8;66(12):2531-2549. Epub 2018 Sep 8.

IRCCS Humanitas, via Manzoni 56, 20089, Rozzano, Italy.

Microglia, brain cells of nonneural origin, orchestrate the inflammatory response to diverse insults, including hypoxia/ischemia or maternal/fetal infection in the perinatal brain. Experimental studies have demonstrated the capacity of microglia to recognize pathogens or damaged cells activating a cytotoxic response that can exacerbate brain damage. However, microglia display an enormous plasticity in their responses to injury and may also promote resolution stages of inflammation and tissue regeneration. Despite the critical role of microglia in brain pathologies, the cellular mechanisms that govern the diverse phenotypes of microglia are just beginning to be defined. Here we review emerging strategies to drive microglia toward beneficial functions, selectively reporting the studies which provide insights into molecular mechanisms underlying the phenotypic switch. A variety of approaches have been proposed which rely on microglia treatment with pharmacological agents, cytokines, lipid messengers, or microRNAs, as well on nutritional approaches or therapies with immunomodulatory cells. Analysis of the molecular mechanisms relevant for microglia reprogramming toward pro-regenerative functions points to a central role of energy metabolism in shaping microglial functions. Manipulation of metabolic pathways may thus provide new therapeutic opportunities to prevent the deleterious effects of inflammatory microglia and to control excessive inflammation in brain disorders.
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http://dx.doi.org/10.1002/glia.23484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585737PMC
December 2018

Differential local tissue permissiveness influences the final fate of GPR17-expressing oligodendrocyte precursors in two distinct models of demyelination.

Glia 2018 05 9;66(5):1118-1130. Epub 2018 Feb 9.

Laboratory of Molecular and Cellular Pharmacology of the Purinergic Transmission, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, Milan, 20133, Italy.

Promoting remyelination is recognized as a novel strategy to foster repair in neurodegenerative demyelinating diseases, such as multiple sclerosis. In this respect, the receptor GPR17, recently emerged as a new target for remyelination, is expressed by early oligodendrocyte precursors (OPCs) and after a certain differentiation stage it has to be downregulated to allow progression to mature myelinating oligodendrocytes. Here, we took advantage of the first inducible GPR17 reporter mouse line (GPR17-iCreER xCAG-eGFP mice) allowing to follow the final fate of GPR17 cells by tamoxifen-induced GFP-labeling to unveil the destiny of these cells in two demyelination models: experimental autoimmune encephalomyelitis (EAE), characterized by marked immune cell activation and inflammation, and cuprizone induced demyelination, where myelin dysfunction is achieved by a toxic insult. In both models, demyelination induced a strong increase of fluorescent GFP cells at damaged areas. However, only in the cuprizone model reacting GFP cells terminally differentiated to mature oligodendrocytes, thus contributing to remyelination. In EAE, GFP cells were blocked at immature stages and never became myelinating oligodendrocytes. We suggest these strikingly distinct fates be due to different permissiveness of the local CNS environment. Based on previously reported GPR17 activation by emergency signals (e.g., Stromal Derived Factor-1), we propose that a marked inflammatory milieu, such as that reproduced in EAE, induces GPR17 overactivation resulting in impaired downregulation, untimely and prolonged permanence in OPCs, leading, in turn, to differentiation blockade. Combined treatments with remyelinating agents and anti-inflammatory drugs may represent new potential adequate strategies to halt neurodegeneration and foster recovery.
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http://dx.doi.org/10.1002/glia.23305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5900886PMC
May 2018

Pathophysiological Role of Purines and Pyrimidines in Neurodevelopment: Unveiling New Pharmacological Approaches to Congenital Brain Diseases.

Front Pharmacol 2017 19;8:941. Epub 2017 Dec 19.

Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy.

In recent years, a substantial body of evidence has emerged demonstrating that purine and pyrimidine synthesis and metabolism play major roles in controlling embryonic and fetal development and organogenesis. Dynamic and time-dependent changes in the expression of purine metabolizing enzymes (such as ectonucleotidases and adenosine deaminase) represent a key checkpoint for the correct sequential generation of the different signaling molecules, that in turn activate their specific membrane receptors. In neurodevelopment, Ca release from radial glia mediated by P2Y purinergic receptors is fundamental to allow neuroblast migration along radial glia processes, and their correct positioning in the different layers of the developing neocortex. Moreover, ATP is involved in the development of synaptic transmission and contributes to the establishment of functional neuronal networks in the developing brain. Additionally, several purinergic receptors (spanning from adenosine to P2X and P2Y receptor subtypes) are differentially expressed by neural stem cells, depending on their maturation stage, and their activation tightly regulates cell proliferation and differentiation to either neurons or glial cells, as well as their correct colonization of the developing telencephalon. The purinergic control of neurodevelopment is not limited to prenatal life, but is maintained in postnatal life, when it plays fundamental roles in controlling oligodendrocyte maturation from precursors and their terminal differentiation to fully myelinating cells. Based on the above-mentioned and other literature evidence, it is now increasingly clear that any defect altering the tight regulation of purinergic transmission and of purine and pyrimidine metabolism during pre- and post-natal brain development may translate into functional deficits, which could be at the basis of severe pathologies characterized by mental retardation or other disturbances. This can occur either at the level of the recruitment and/or signaling of specific nucleotide or nucleoside receptors or through genetic alterations in key steps of the purine salvage pathway. In this review, we have provided a critical analysis of what is currently known on the pathophysiological role of purines and pyrimidines during brain development with the aim of unveiling new future strategies for pharmacological intervention in different neurodevelopmental disorders.
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http://dx.doi.org/10.3389/fphar.2017.00941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770749PMC
December 2017

High Yield of Adult Oligodendrocyte Lineage Cells Obtained from Meningeal Biopsy.

Front Pharmacol 2017 12;8:703. Epub 2017 Oct 12.

Section of Pharmacology, Department of Diagnostics and Public Health, University of Verona, Verona, Italy.

Oligodendrocyte loss can lead to cognitive and motor deficits. Current remyelinating therapeutic strategies imply either modulation of endogenous oligodendrocyte precursors or transplantation of expanded oligodendrocytes. Cell therapy, however, still lacks identification of an adequate source of oligodendrocyte present in adulthood and able to efficiently produce transplantable cells. Recently, a neural stem cell-like population has been identified in meninges. We developed a protocol to obtain high yield of oligodendrocyte lineage cells from one single biopsy of adult rat meningeal tissue. From 1 cm of adult rat spinal cord meninges, we efficiently expanded a homogenous culture of 10 millions of meningeal-derived oligodendrocyte lineage cells in a short period of time (approximately 4 weeks). Meningeal-derived oligodendrocyte lineage cells show typical mature oligodendrocyte morphology and express specific oligodendrocyte markers, such as galactosylceramidase and myelin basic protein. Moreover, when transplanted in a chemically demyelinated spinal cord model, meningeal-derived oligodendrocyte lineage cells display -remyelinating potential. This oligodendrocyte lineage cell population derives from an accessible and adult source, being therefore a promising candidate for autologous cell therapy of demyelinating diseases. In addition, the described method to differentiate meningeal-derived neural stem cells into oligodendrocyte lineage cells may represent a valid model to dissect oligodendrocyte differentiation and to screen for drugs capable to promote oligodendrocyte regeneration.
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http://dx.doi.org/10.3389/fphar.2017.00703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643910PMC
October 2017

Pharmacological Properties and Biological Functions of the GPR17 Receptor, a Potential Target for Neuro-Regenerative Medicine.

Adv Exp Med Biol 2017 ;1051:169-192

Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Via Balzaretti 9, 20133, Milan, Italy.

In 2006, cells heterologously expressing the "orphan" receptor GPR17 were shown to acquire responses to both uracil nucleotides and cysteinyl-leukotrienes, two families of signaling molecules accumulating in brain or heart as a result of hypoxic/traumatic injuries. In subsequent years, evidence of GPR17 key role in oligodendrogenesis and myelination has highlighted it as a "model receptor" for new therapies in demyelinating and neurodegenerative diseases. The apparently contrasting evidence in the literature about the role of GPR17 in promoting or inhibiting myelination can be due to its transient expression in the intermediate stages of differentiation, exerting a pro-differentiating function in early oligodendrocyte precursor cells (OPCs), and an inhibitory role in late stage maturing cells. Meanwhile, several papers extended the initial data on GPR17 pharmacology, highlighting a "promiscuous" behavior of this receptor; indeed, GPR17 is able to respond to other emergency signals like oxysterols or the pro-inflammatory cytokine SDF-1, underlying GPR17 ability to adapt its responses to changes of the surrounding extracellular milieu, including damage conditions. Here, we analyze the available literature on GPR17, in an attempt to summarize its emerging biological roles and pharmacological properties.
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http://dx.doi.org/10.1007/5584_2017_92DOI Listing
June 2018

The role of oligodendrocyte precursor cells expressing the GPR17 receptor in brain remodeling after stroke.

Cell Death Dis 2017 06 8;8(6):e2871. Epub 2017 Jun 8.

Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy.

Following stroke-induced neuronal damage, quiescent oligodendrocyte precursors (OPCs) are activated to proliferate and later to differentiate to myelin-producing cells. GPR17, a receptor transiently expressed on early OPCs, has emerged as a target to implement stroke repair through stimulation of OPC maturation. However, being GPR17 completely downregulated in myelin-producing oligodendrocytes, its actual role in determining the final fate of OPCs after cerebral ischemia is still uncertain. Here, to univocally define the spatiotemporal changes and final fate of GPR17-expressing OPCs, we induced ischemia by middle cerebral artery occlusion (MCAo) in reporter GPR17iCreER:CAG-eGreen florescent protein (GFP) mice, in which, upon tamoxifen treatment, cells expressing GPR17 become green and traceable for their entire life. Starting from 3 days and up to 2 weeks after MCAo, GFP cells markedly accumulated in regions surrounding the ischemic lesion; several of them proliferated, as shown by co-labeling of the DNA synthesis marker 5-Bromo-2'-deoxyuridine (BrdU). Almost all GFP/BrdU cells expressed the OPC early marker neural/glial antigen 2 (NG2), indicating that they were still precursors. Accumulation of GFP cells was also because of OPC recruitment from surrounding areas, as suggested in vivo by acquisition of typical features of migrating OPCs, shown in vitro in presence of the chemoattractant PDGF-AA and confirmed by transplantation of GFP-OPCs in wild-type MCAo mice. Eight weeks after MCAo, only some of these precociously recruited cells had undergone maturation as shown by NG2 loss and acquisition of mature myelinating markers like GSTpi. A pool of recruited GFP-OPCs was kept at a precursor stage to likely make it available for further insults. Thus, very early after ischemia, GFP-OPCs proliferate and migrate toward the lesion; however, most of these cells remain undifferentiated, suggesting functional roles other than myelination.
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http://dx.doi.org/10.1038/cddis.2017.256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5520912PMC
June 2017

Intertwining extracellular nucleotides and their receptors with Ca2+ in determining adult neural stem cell survival, proliferation and final fate.

Philos Trans R Soc Lond B Biol Sci 2016 08;371(1700)

Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, 20133 Milan, Italy

In the central nervous system (CNS), during both brain and spinal cord development, purinergic and pyrimidinergic signalling molecules (ATP, UTP and adenosine) act synergistically with peptidic growth factors in regulating the synchronized proliferation and final specification of multipotent neural stem cells (NSCs) to neurons, astrocytes or oligodendrocytes, the myelin-forming cells. Some NSCs still persist throughout adulthood in both specific 'neurogenic' areas and in brain and spinal cord parenchyma, retaining the potentiality to generate all the three main types of adult CNS cells. Once CNS anatomical structures are defined, purinergic molecules participate in calcium-dependent neuron-to-glia communication and also control the behaviour of adult NSCs. After development, some purinergic mechanisms are silenced, but can be resumed after injury, suggesting a role for purinergic signalling in regeneration and self-repair also via the reactivation of adult NSCs. In this respect, at least three different types of adult NSCs participate in the response of the adult brain and spinal cord to insults: stem-like cells residing in classical neurogenic niches, in particular, in the ventricular-subventricular zone (V-SVZ), parenchymal oligodendrocyte precursor cells (OPCs, also known as NG2-glia) and parenchymal injury-activated astrocytes (reactive astrocytes). Here, we shall review and discuss the purinergic regulation of these three main adult NSCs, with particular focus on how and to what extent modulation of intracellular calcium levels by purinoceptors is mandatory to determine their survival, proliferation and final fate.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.
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http://dx.doi.org/10.1098/rstb.2015.0433DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938032PMC
August 2016

CNS remyelination as a novel reparative approach to neurodegenerative diseases: The roles of purinergic signaling and the P2Y-like receptor GPR17.

Neuropharmacology 2016 05 8;104:82-93. Epub 2015 Oct 8.

Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Via Balzaretti 9, 20133 Milan, Italy. Electronic address:

Oligodendrocytes are the myelin-forming cells in the CNS. They enwrap axons, thus permitting fast impulse transmission and exerting trophic actions on neurons. Demyelination accompanied by neurological deficit is a rather frequent condition that is not only associated with multiple sclerosis but has been also recognized in several other neurodegenerative diseases, including brain trauma and stroke, Alzheimer's disease and amyotrophic lateral sclerosis. Recently, alterations of myelin function have been also reported in neuropsychiatric diseases, like depression and autism. Highly relevant for therapeutic purposes, oligodendrocyte precursor cells (OPCs) still persist in the adult brain and spinal cord. These cells are normally rather quiescent, but under specific circumstances, they can be stimulated to undergo differentiation and generate mature myelinating oligodendrocytes. Thus, approaches aimed at restoring myelin integrity and at fostering a correct oligodendrocyte function are now viewed as novel therapeutic opportunities for both neurodegenerative and neuropsychiatric diseases. Both OPCs and mature oligodendrocytes express purinergic receptors. For some of these receptors, expression is restricted at specific differentiation stages, suggesting key roles in OPCs maturation and myelination. Some of these receptors are altered under demyelinating conditions, suggesting that their dysregulation may contribute to disease development and could represent adequate new targets for remyelinating therapies. Here, we shall describe the current literature available on all these receptors, with special emphasis on the P2Y-like GPR17 receptor, that represents one of the most studied receptor subtypes in these cells. 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.2015.10.005DOI Listing
May 2016

The ubiquitin ligase Mdm2 controls oligodendrocyte maturation by intertwining mTOR with G protein-coupled receptor kinase 2 in the regulation of GPR17 receptor desensitization.

Glia 2015 Dec 31;63(12):2327-39. Epub 2015 Jul 31.

Department of Pharmacological and Biomolecular Sciences, Università Degli Studi Di Milano, Milan, Italy.

During oligodendrocyte precursor cell (OPC) differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impair remyelination under demyelinating conditions. After the immature oligodendrocyte stage, to enable cells to complete maturation, GPR17 is physiologically down-regulated via phosphorylation/desensitization by G protein-coupled receptor kinases (GRKs); conversely, GRKs are regulated by the "mammalian target of rapamycin" mTOR. However, how GRKs and mTOR are connected to each other in modulating GPR17 function and oligodendrogenesis has remained elusive. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of the onco-suppressor p53 protein. In maturing OPCs, both rapamycin and Nutlin-3, a small molecule inhibitor of Mdm2-p53 interactions, increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. Thus, Mdm2 intertwines mTOR with GRK2 in regulating GPR17 and oligodendrogenesis and represents a novel actor in myelination.
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http://dx.doi.org/10.1002/glia.22896DOI Listing
December 2015

Microglia is a key player in the reduction of stroke damage promoted by the new antithrombotic agent ticagrelor.

J Cereb Blood Flow Metab 2014 Jun 19;34(6):979-88. Epub 2014 Mar 19.

1] Department of Pharmacological and Biomolecular Sciences, University of Milan, Milan, Italy [2] Centro Cardiologico Monzino IRCCS, Milan, Italy.

The ADP-responsive P2Y12 receptor is expressed on both platelets and microglia. Clinical data show that ticagrelor, a direct-acting, reversibly binding P2Y12-receptor antagonist, reduces total cardiovascular events, including stroke. In our present study, we investigated the expression of P2Y12 receptors and the effects of ticagrelor on brain injury in Sprague-Dawley rats subjected to a permanent middle cerebral artery occlusion (MCAo). Rats were treated per os with ticagrelor 3 mg/kg or vehicle at 10 minutes, 22, and 36 hours after MCAo and killed after 48 hours. Immunofluorescence analysis showed an ischemia-related modulation of the P2Y12 receptor, which is constitutively expressed in Iba1(+) resting microglia. After MCAo, activated microglia was mainly concentrated around the lesion, with fewer cells present inside the ischemic core. Ticagrelor significantly attenuated the evolution of ischemic damage-evaluated by magnetic resonance imaging (MRI) at 2, 24, and 48 hours after MCAo-, the number of infiltrating cells expressing the microglia/monocyte marker ED-1, the cerebral expression of proinflammatory mediators (interleukin 1 (IL-1), monocyte chemoattractant protein 1 (MCP-1), nitric oxide synthase (iNOS)) and the associated neurologic impairment. In transgenic fluorescent reporter CX3CR1-green fluorescent protein (GFP) mice, 72 hours after MCAo, ticagrelor markedly reduced GFP(+) microglia and both early and late infiltrating blood-borne cells. Finally, in primary cultured microglia, ticagrelor fully inhibited ADP-induced chemotaxis (P<0.01). Our results show that ticagrelor is protective against ischemia-induced cerebral injury and this effect is mediated, at least partly, by inhibition of P2Y12-mediated microglia activation and chemotaxis.
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http://dx.doi.org/10.1038/jcbfm.2014.45DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050242PMC
June 2014

Does GRK-β arrestin machinery work as a "switch on" for GPR17-mediated activation of intracellular signaling pathways?

Cell Signal 2014 Jun 5;26(6):1310-25. Epub 2014 Mar 5.

Department of Pharmacy, University of Pisa, 56126 Pisa, Italy. Electronic address:

During oligodendrocyte-precursor cell (OPC) differentiation program, an impairment in the regulatory mechanisms controlling GPR17 spatio-temporal expression and functional activity has been suggested to contribute to defective OPC maturation, a crucial event in the pathogenesis of multiple sclerosis. GRK-β arrestin machinery is the primary actor in the control of G-protein coupled receptor (GPCR) functional responses and changes in these regulatory protein activities have been demonstrated in several immune/inflammatory diseases. Herein, in order to shed light on the molecular mechanisms controlling GPR17 regulatory events during cell differentiation, the role of GRK/β-arrestin machinery in receptor desensitization and signal transduction was investigated, in transfected cells and primary OPC. Following cell treatment with the two classes of purinergic and cysteinyl-leukotriene (cysLT) ligands, different GRK isoforms were recruited to regulate GPR17 functional responses. CysLT-mediated receptor desensitization mainly involved GRK2; this kinase, via a G protein-dependent mechanism, promoted a transient binding of the receptor to β-arrestins, rapid ERK phosphorylation and sustained nuclear CREB activation. Furthermore, GRK2, whose expression parallels that of the receptor during differentiation process, appeared to be crucial to induce cysLT-mediated maturation of OPCs. On the other hand, purinergic ligand exclusively recruited the GRK5 subtype, and induced, via a G protein-independent/β-arrestin-dependent mechanism, a receptor/β-arrestin stable association, slower and sustained ERK stimulation and marginal CREB activation. These results show that purinergic and cysLT ligands, through the recruitment of specific GRK isoforms, address distinct intracellular pathways, most likely reinforcing the same final response. The identification of these mechanisms and players controlling GPR17 responses during OPC differentiation could be useful to identify new targets in demyelination diseases and to develop new therapeutical strategies.
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http://dx.doi.org/10.1016/j.cellsig.2014.02.016DOI Listing
June 2014

Changes of the GPR17 receptor, a new target for neurorepair, in neurons and glial cells in patients with traumatic brain injury.

Purinergic Signal 2013 Sep 26;9(3):451-62. Epub 2013 Jun 26.

Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Leipzig, Germany.

Unveiling the mechanisms participating in the damage and repair of traumatic brain injury (TBI) is fundamental to develop new therapies. The P2Y-like GPR17 receptor has recently emerged as a sensor of damage and a key actor in lesion remodeling/repair in the rodent brain, but its role in humans is totally unknown. Here, we characterized GPR17 expression in brain specimens from seven intensive care unit TBI patients undergoing neurosurgery for contusion removal and from 28 autoptic TBI cases (and 10 control subjects of matched age and gender) of two university hospitals. In both neurosurgery and autoptic samples, GPR17 expression was strong inside the contused core and progressively declined distally according to a spatio-temporal gradient. Inside and around the core, GPR17 labeled dying neurons, reactive astrocytes, and activated microglia/macrophages. In peri-contused parenchyma, GPR17 decorated oligodendrocyte precursor cells (OPCs) some of which had proliferated, indicating re-myelination attempts. In autoptic cases, GPR17 expression positively correlated with death for intracranial complications and negatively correlated with patients' post-traumatic survival. Data indicate lesion-specific sequential involvement of GPR17 in the (a) death of irreversibly damaged neurons, (b) activation of microglia/macrophages remodeling the lesion, and (c) activation/proliferation of multipotent parenchymal progenitors (both reactive astrocytes and OPCs) starting repair processes. Data validate GPR17 as a target for neurorepair and are particularly relevant to setting up new therapies for TBI patients.
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http://dx.doi.org/10.1007/s11302-013-9366-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3757149PMC
September 2013

Cellular and molecular characterization of multipolar Map5-expressing cells: a subset of newly generated, stage-specific parenchymal cells in the mammalian central nervous system.

PLoS One 2013 7;8(5):e63258. Epub 2013 May 7.

Neuroscience Institute Cavalieri Ottolenghi and Department of Veterinary Sciences, University of Turin, Turin, Italy.

Although extremely interesting in adult neuro-glio-genesis and promising as an endogenous source for repair, parenchymal progenitors remain largely obscure in their identity and physiology, due to a scarce availability of stage-specific markers. What appears difficult is the distinction between real cell populations and various differentiation stages of the same population. Here we focused on a subset of multipolar, polydendrocyte-like cells (mMap5 cells) expressing the microtubule associated protein 5 (Map5), which is known to be present in most neurons. We characterized the morphology, phenotype, regional distribution, proliferative dynamics, and stage-specific marker expression of these cells in the rabbit and mouse CNS, also assessing their existence in other mammalian species. mMap5 cells were never found to co-express the Ng2 antigen. They appear to be a population of glial cells sharing features but also differences with Ng2+progenitor cells. We show that mMap5 cells are newly generated, postmitotic parenchymal elements of the oligodendroglial lineage, thus being a stage-specific population of polydendrocytes. Finally, we report that the number of mMap5 cells, although reduced within the brain of adult/old animals, can increase in neurodegenerative and traumatic conditions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0063258PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647045PMC
December 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

The regulated expression, intracellular trafficking, and membrane recycling of the P2Y-like receptor GPR17 in Oli-neu oligodendroglial cells.

J Biol Chem 2013 Feb 3;288(7):5241-56. Epub 2013 Jan 3.

Consiglio Nazionale delle Ricerche-Institute of Neuroscience, Department of Medical Biotechnologies and Translational Medicine, University of Milan, Milan 20129, Italy.

GPR17 is a G-protein-coupled receptor that is activated by two classes of molecules: uracil-nucleotides and cysteinyl-leukotrienes. GPR17 is required for initiating the differentiation of oligodendrocyte precursors but has to be down-regulated to allow cells to undergo terminal maturation. Although a great deal has been learned about GPR17 expression and signaling, no information is currently available about the trafficking of native receptors after the exposure of differentiating oligodendrocytes to endogenous agonists. Here, we demonstrate that neuron-conditioned medium induces the transcriptionally mediated, time-regulated expression of GPR17 in Oli-neu, an oligodendrocyte precursor cell line, making these cells suitable for studying the endocytic traffic of the native receptor. Agonist-induced internalization, intracellular trafficking, and membrane recycling of GPR17 were analyzed by biochemical and immunofluorescence assays using an ad hoc-developed antibody against the extracellular N-terminal of GPR17. Both UDP-glucose and LTD(4) increased GPR17 internalization, although with different efficiency. At early time points, internalized GPR17 co-localized with transferrin receptor, whereas at later times it partially co-localized with the lysosomal marker Lamp1, suggesting that a portion of GPR17 is targeted to lysosomes upon ligand binding. An analysis of receptor recycling and degradation demonstrated that a significant aliquot of GPR17 is recycled to the cell surface. Furthermore, internalized GPR17 displayed a co-localization with the marker of the "short loop" recycling endosomes, Rab4, while showing very minor co-localization with the "long loop" recycling marker, Rab11. Our results provide the first data on the agonist-induced trafficking of native GPR17 in oligodendroglial cells and may have implications for both physiological and pathological myelination.
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http://dx.doi.org/10.1074/jbc.M112.404996DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3576128PMC
February 2013

Purinergic trophic signalling in glial cells: functional effects and modulation of cell proliferation, differentiation, and death.

Purinergic Signal 2012 Sep 12;8(3):539-57. Epub 2012 Apr 12.

Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological Sciences, Università degli Studi di Milano, via Balzaretti, 9-Milan, 20133, Italy.

In the last decades, the discovery that glial cells do not only fill in the empty space among neurons or furnish them with trophic support but are rather essential participants to the various activities of the central and peripheral nervous system has fostered the search for the signalling pathways controlling their functions. Since the early 1990s, purines were foreseen as some of the most promising candidate molecules. Originally just a hypothesis, this has become a certainty as experimental evidence accumulated over years, as demonstrated by the exponentially growing number of articles related to the role of extracellular nucleotides and nucleosides in controlling glial cell functions. Indeed, as new functions for already known glial cells (for example, the ability of parenchymal astrocytes to behave as stem cells) or new subtypes of glial cells (for example, NG2(+) cells, also called polydendrocytes) are discovered also, new actions and new targets for the purinergic system are identified. Thus, glial purinergic receptors have emerged as new possible pharmacological targets for various acute and chronic pathologies, such as stroke, traumatic brain and spinal cord injury, demyelinating diseases, trigeminal pain and migraine, and retinopathies. In this article, we will summarize the most important and promising actions mediated by extracellular purines and pyrimidines in controlling the functions, survival, and differentiation of the various "classical" types of glial cells (i.e., astrocytes, oligodendrocytes, microglial cells, Müller cells, satellite glial cells, and enteric glial cells) but also of some rather new members of the family (e.g., polydendrocytes) and of other cells somehow related to glial cells (e.g., pericytes and spinal cord ependymal cells).
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http://dx.doi.org/10.1007/s11302-012-9310-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360088PMC
September 2012