Elisabetta Bonfanti

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

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.

GPR17 expressing NG2-Glia: Oligodendrocyte progenitors serving as a reserve pool after injury.

In the adult brain NG2‐glia continuously generate mature, myelinating oligodendrocytes. To which extent the differentiation process is common to all NG2‐glia and whether distinct pools are recruited for repair under physiological and pathological conditions still needs clarification. Here, we aimed at investigating the differentiation potential of adult NG2‐glia that specifically express the G‐protein coupled receptor 17 (GPR17), a membrane receptor that regulates the differentiation of these cells at postnatal stages. To this aim, we generated the first BAC transgenic GPR17‐iCreERT2 mouse line for fate mapping studies. In these mice, under physiological conditions, GPR17+ cells —in contrast to GPR17‐ NG2‐glia— did not differentiate within 3 months, a peculiarity that was overcome after cerebral damage induced by acute injury or ischemia. After these insults, GPR17+ NG2‐glia rapidly reacted to the damage and underwent maturation, suggesting that they represent a ‘reserve pool’ of adult progenitors maintained for repair purposes. GLIA 2016;64:287–299

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

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.

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

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. GLIA 2015;63:2327–2339

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

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 GPR17iCreERT2: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.

High Yield of Adult Oligodendrocyte Lineage Cells Obtained from Meningeal Biopsy

Oligodendrocyte loss can lead to cognitive and motor deficits. Current remyelinating therapeutic strategies imply either modulation of endogenous oligodendrocyte precursors or transplantation of in vitro 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 cm2 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 in vivo-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 in vitro model to dissect oligodendrocyte differentiation and to screen for drugs capable to promote oligodendrocyte regeneration.

Extracellular purine and pyrimidine nucleotides as local extrinsic regulators of adult neural progenitor cells in the diseased CNS

Autism is a severe neurodevelopmental disorder characterized by pervasive impairment in social interaction, deficits in verbal and nonverbal communication, and restricted, repetitive, and stereotyped patterns of behavior, interests, and activities, with a four times higher incidence in boys than girls and accompanied frequently by neurobehavioral core symptoms and immunological derangements, including: aberrant sensitivity to sensory stimulation, anxiety, and decreased cellular immune capacity (American Psychiatric Association, 1994). More current studies have provided evidence suggesting that autism is a pathophysiological process arising from the interaction of an early environmental insult with a genetic predisposition for the disease to the neuroimmune dysregulation, in all cases becoming as biomolecular markers associated in a high rate to autism spectrum disorder and to reinforce the aberrant autoimmune reaction and neuroimmune interaction in this disorder. Current aspects on in connect will be considered in this presentation. This paper will show a review on the most current aspects regarding the etiology and neuropathology in autism from the molecular biomarkers associated, remarking the aspects on the aberrant neuroimmune interaction explored from biomolecular markers.