G protein-coupled receptor 17 is regulated by WNT pathway during oligodendrocyte precursor cell differentiation.

G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to achieve fully-myelinating cells. However, whether and how these two systems are related to each other is still unknown. Of interest, both factors are dysregulated in developing and adult brain diseases, including white matter injury and cancer, making the understanding of their reciprocal interactions of potential importance for identifying new targets and strategies for myelin repair. Here, by a combined pharmacological and biotechnological approach, we examined regulatory mechanisms linking WNT signaling to GPR17 expression in OLs. We first analyzed the relative expression of mRNAs encoding for GPR17 and the T cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF) transcription factors of the canonical WNT/ß-CATENIN pathway, in PDGFRα+ and O4+ OLs during mouse post-natal development. In O4+ cells, Gpr17 mRNA level peaked at post-natal day 14 and then decreased concomitantly to the physiological uprise of WNT tone, as shown by increased Lef1 mRNA level. The link between WNT signaling and GPR17 expression was further reinforced in vitro in primary PDGFRα+ cells and in Oli-neu cells. High WNT tone impaired OL differentiation and drastically reduced GPR17 mRNA and protein levels. In Oli-neu cells, WNT/ß-CATENIN activation repressed Gpr17 promoter activity through both putative WNT response elements (WRE) and upregulation of the inhibitor of DNA-binding protein 2 (Id2). We conclude that the WNT pathway influences OL maturation by repressing GPR17, which could have implications in pathologies characterized by dysregulations of the OL lineage including multiple sclerosis and oligodendroglioma.

Altered Purinergic Signaling in Neurodevelopmental Disorders: Focus on P2 Receptors.

With the umbrella term 'neurodevelopmental disorders' (NDDs) we refer to a plethora of congenital pathological conditions generally connected with cognitive, social behavior, and sensory/motor alterations. Among the possible causes, gestational and perinatal insults have been demonstrated to interfere with the physiological processes necessary for the proper development of fetal brain cytoarchitecture and functionality. In recent years, several genetic disorders caused by mutations in key enzymes involved in purine metabolism have been associated with autism-like behavioral outcomes. Further analysis revealed dysregulated purine and pyrimidine levels in the biofluids of subjects with other NDDs. Moreover, the pharmacological blockade of specific purinergic pathways reversed the cognitive and behavioral defects caused by maternal immune activation, a validated and now extensively used rodent model for NDDs. Furthermore, Fragile X and Rett syndrome transgenic animal models as well as models of premature birth, have been successfully utilized to investigate purinergic signaling as a potential pharmacological target for these diseases. In this review, we examine results on the role of the P2 receptor signaling in the etiopathogenesis of NDDs. On this basis, we discuss how this evidence could be exploited to develop more receptor-specific ligands for future therapeutic interventions and novel prognostic markers for the early detection of these conditions.

Rewiring of Glucose and Lipid Metabolism Induced by G Protein-Coupled Receptor 17 Silencing Enables the Transition of Oligodendrocyte Progenitors to Myelinating Cells.

In the mature central nervous system (CNS), oligodendrocytes (OLs) provide support and insulation to axons thanks to the production of a myelin sheath. During their maturation to myelinating cells, OLs require energy and building blocks for lipids, which implies a great investment of energy fuels and molecular sources of carbon. The oligodendroglial G protein-coupled receptor 17 (GPR17) has emerged as a key player in OL maturation; it reaches maximal expression in pre-OLs, but then it has to be internalized to allow terminal maturation. In this study, we aim at elucidating the role of physiological GPR17 downregulation in OL metabolism by applying transcriptomics, metabolomics and lipidomics on differentiating OLs. After GPR17 silencing, we found a significant increase in mature OL markers and alteration of several genes involved in glucose metabolism and lipid biosynthesis. We also observed an increased release of lactate, which is partially responsible for the maturation boost induced by GPR17 downregulation. Concomitantly, GPR17 depletion also changed the kinetics of specific myelin lipid classes. Globally, this study unveils a functional link between GPR17 expression, lactate release and myelin composition, and suggests that innovative interventions targeting GPR17 may help to foster endogenous myelination in demyelinating diseases.

Novel in vitro Experimental Approaches to Study Myelination and Remyelination in the Central Nervous System.

Myelin is the lipidic insulating structure enwrapping axons and allowing fast saltatory nerve conduction. In the central nervous system, myelin sheath is the result of the complex packaging of multilamellar extensions of oligodendrocyte (OL) membranes. Before reaching myelinating capabilities, OLs undergo a very precise program of differentiation and maturation that starts from OL precursor cells (OPCs). In the last 20 years, the biology of OPCs and their behavior under pathological conditions have been studied through several experimental models. When co-cultured with neurons, OPCs undergo terminal maturation and produce myelin tracts around axons, allowing to investigate myelination in response to exogenous stimuli in a very simple in vitro system. On the other hand, in vivo models more closely reproducing some of the features of human pathophysiology enabled to assess the consequences of demyelination and the molecular mechanisms of remyelination, and they are often used to validate the effect of pharmacological agents. However, they are very complex, and not suitable for large scale drug discovery screening. Recent advances in cell reprogramming, biophysics and bioengineering have allowed impressive improvements in the methodological approaches to study brain physiology and myelination. Rat and mouse OPCs can be replaced by human OPCs obtained by induced pluripotent stem cells (iPSCs) derived from healthy or diseased individuals, thus offering unprecedented possibilities for personalized disease modeling and treatment. OPCs and neural cells can be also artificially assembled, using 3D-printed culture chambers and biomaterial scaffolds, which allow modeling cell-to-cell interactions in a highly controlled manner. Interestingly, scaffold stiffness can be adopted to reproduce the mechanosensory properties assumed by tissues in physiological or pathological conditions. Moreover, the recent development of iPSC-derived 3D brain cultures, called organoids, has made it possible to study key aspects of embryonic brain development, such as neuronal differentiation, maturation and network formation in temporal dynamics that are inaccessible to traditional in vitro cultures. Despite the huge potential of organoids, their application to myelination studies is still in its infancy. In this review, we shall summarize the novel most relevant experimental approaches and their implications for the identification of remyelinating agents for human diseases such as multiple sclerosis.

Involvement of GPR17 in Neuronal Fibre Outgrowth.

Characterization of new pharmacological targets is a promising approach in research of neurorepair mechanisms. The G protein-coupled receptor 17 (GPR17) has recently been proposed as an interesting pharmacological target, e.g., in neuroregenerative processes. Using the well-established ex vivo model of organotypic slice co-cultures of the mesocortical dopaminergic system (prefrontal cortex (PFC) and substantia nigra/ventral tegmental area (SN/VTA) complex), the influence of GPR17 ligands on neurite outgrowth from SN/VTA to the PFC was investigated. The growth-promoting effects of Montelukast (MTK; GPR17- and cysteinyl-leukotriene receptor antagonist), the glial cell line-derived neurotrophic factor (GDNF) and of two potent, selective GPR17 agonists (PSB-16484 and PSB-16282) were characterized. Treatment with MTK resulted in a significant increase in mean neurite density, comparable with the effects of GDNF. The combination of MTK and GPR17 agonist PSB-16484 significantly inhibited neuronal growth. qPCR studies revealed an MTK-induced elevated mRNA-expression of genes relevant for neuronal growth. Immunofluorescence labelling showed a marked expression of GPR17 on NG2-positive glia. Western blot and RT-qPCR analysis of untreated cultures suggest a time-dependent, injury-induced stimulation of GPR17. In conclusion, MTK was identified as a stimulator of neurite fibre outgrowth, mediating its effects through GPR17, highlighting GPR17 as an interesting therapeutic target in neuronal regeneration.

The Distribution of GPR17-Expressing Cells Correlates with White Matter Inflammation Status in Brain Tissues of Multiple Sclerosis Patients.

In multiple sclerosis (MS), oligodendrocyte precursor cells (OPCs) are recruited to the site of injury to remyelinate damaged axons; however, in patients this process is often ineffective due to defects in OPC maturation. The membrane receptor GPR17 timely regulates the early stages of OPC differentiation; however, after reaching its highest levels in immature oligodendrocytes, it has to be downregulated to allow terminal maturation. Since, in several animal models of disease GPR17 is upregulated, the aim of this work was to characterize GPR17 alterations in MS patients. We developed immunohistochemistry and immunofluorescence procedures for the detection of GPR17 in human tissues and stained post-mortem MS brain lesions from patients with secondary progressive MS and control subjects. The inflammatory activity in each lesion was evaluated by immunohistochemistry for the myelin protein MOG and the HLA antigen to classify them as active, chronic inactive or chronic active. Hence, we assessed the distribution of GPR17-positive cells in these lesions compared to normal appearing white matter (NAWM) and white matter (WM) of control subjects. Our data have shown a marked increase of GPR17-expressing oligodendroglial cells accumulating at NAWM, in which moderate inflammation was also found. Furthermore, we identified two distinct subpopulations of GPR17-expressing oligodendroglial cells, characterized by either ramified or rounded morphology, that differently populate the WM of healthy controls and MS patients. We concluded that the coordinated presence of GPR17 in OPCs at the lesion sites and inflamed NAWM areas suggests that GPR17 could be exploited to support endogenous remyelination through advanced pharmacological approaches.

Functional genomic analyses highlight a shift in Gpr17-regulated cellular processes in oligodendrocyte progenitor cells and underlying myelin dysregulation in the aged mouse cerebrum.

Brain ageing is characterised by a decline in neuronal function and associated cognitive deficits. There is increasing evidence that myelin disruption is an important factor that contributes to the age-related loss of brain plasticity and repair responses. In the brain, myelin is produced by oligodendrocytes, which are generated throughout life by oligodendrocyte progenitor cells (OPCs). Currently, a leading hypothesis points to ageing as a major reason for the ultimate breakdown of remyelination in Multiple Sclerosis (MS). However, an incomplete understanding of the cellular and molecular processes underlying brain ageing hinders the development of regenerative strategies. Here, our combined systems biology and neurobiological approach demonstrate that oligodendroglial and myelin genes are amongst the most altered in the ageing mouse cerebrum. This was underscored by the identification of causal links between signalling pathways and their downstream transcriptional networks that define oligodendroglial disruption in ageing. The results highlighted that the G-protein coupled receptor Gpr17 is central to the disruption of OPCs in ageing and this was confirmed by genetic fate-mapping and cellular analyses. Finally, we used systems biology strategies to identify therapeutic agents that rejuvenate OPCs and restore myelination in age-related neuropathological contexts.

The history of the Purine Club: a tribute to Prof. Geoffrey Burnstock.

The international purinergic scientific community has lost its pioneer. Geoffrey Burnstock, born on the 10th of May 1929 in London, died on the 2nd of June 2020, aged 91, in Melbourne (Australia). Geoff was one of the most highly regarded scientists of his generation. In the 1960s and 1970s, he developed a radical and somehow heretical new theory and opened an entire new field of science, signalling via extracellular nucleotides (the "purinergic theory"), which revolutionized our understanding of how cells communicate between each other. Initially, his unconventional theory found a lot of resistance in the scientific community. Once, one scientist even threatened to devote his entire life to disproving Burnstock's theory. Undeterred, Geoff went further on, and continued to accumulate evidence in favour of his hypothesis, and led the field ever since. He struggled to attract new scientists to this new field of research and, in the early 1990s, due to new molecular biology techniques making it possible to isolate and identify cell surface receptors for ATP and its breakdown product adenosine, did evidence emerge that eventually convinced the doubters. The number of spontaneous obituaries and messages honouring Geoff's memory that have appeared on specialized Journals and in the public press throughout the world since last June indicates that many people are clearly affected by his death. Besides being a rigorous, ethical and extremely brilliant scientist, Geoff was an extraordinary human being, always eager to collaborate and share data, never jealous of his findings and capable of learning things even from young people. He was known for his enthusiasm, empathy and ability to motivate young scientists and promote their careers. After the establishment of the Purine Club back in the 1990s, numerous Purine Club Chapters have been formed around the world with Geoff's help and encouragement. He has obviously also been the inspirator and founder of our Journal, Purinergic Signalling (PUSI). For this reason, Charles Kennedy, the current Editor of the Journal, and myself thought that it might be nice to invite representatives from all known Purine Clubs to send a few notes to be published in PUSI on the history of their club and how Geoff inspired, aided or supported them. Here, I have collected all their contributions and I share with the entire purinergic community my personal memories on how the Purine Club was born and developed thanks to the invaluable mentoring of Geoffrey Burnstock. I apologize in advance if I am missing some information or forgot to mention somebody, and I strongly encourage all readers to submit memories and additional information that I shall gather for future writing. Keeping alive the history of how the field developed will be the best tribute that we can play to celebrate Geoff's work along the years.

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

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-iCreERT2: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.

Pathway-Focused Profiling of Oligodendrocytes Over-Expressing miR-125a-3p Reveals Alteration of Wnt and Cell-to-Cell Signaling.

MicroRNAs are small post-transcriptional regulators that modulate gene expression by directly interacting with their target transcripts. Since the interaction between miRNAs and target mRNAs does not require a perfect match, one single miRNA can influence the expression of several genes and lead to a very broad array of functional consequences. Recently, we identified miR-125a-3p as a new regulator of oligodendrocyte development, showing that its over-expression is associated to impaired oligodendrocyte maturation. However, whether and how miR-125a-3p over-expression is causally related to oligodendrocyte maturation is still obscure, as well as the pathways responsible for this effect. To shed light on this issue and to identify the underlying molecular mechanisms, we determined the transcriptomic profile of miR-125a-3p over-expressing oligodendrocytes and, by means of two complementary bioinformatic approaches, we have identified pathways and biological processes consistently modulated by miR-125a-3p alteration. This analysis showed that miR-125a-3p is involved in the regulation of cell-cell interactions and Wnt signaling. By means of pathway-focused PCR arrays, we confirmed that miR-125a-3p induces changes in the expression of several genes encoding for adhesion molecules and gap junctions, which play key roles in oligodendrocytes after exposure to pathological demyelinating stimuli. Moreover, the expression changes of different Wnt targets suggest an over-activation of this pathway. Globally, our studies show that miR-125a-3p over-expression can alter signaling pathways and biological processes essential for myelin formation in oligodendrocytes, suggesting that alteration of miR-125a-3p levels may contribute to impairing oligodendrocyte maturation in demyelinating diseases.

Perspectives on Geoff Burnstock as researcher, teacher and friend.

Geoffrey Burnstock, one of the most talented and brilliant scientists of his generation, was born on the 10th of May 1929 in London and died on the 2ndof June 2020, aged 91, in Melbourne (Australia). Geoffrey Burnstock started his research studies with an interest in the nerves controlling the guts of guinea pigs, and discovered a completely unexpected and ubiquitous signalling system mediated via extracellular nucleotides (the "purinergic theory"), which revolutionized our understanding of how cells communicate between each other. He made the highly controversial discovery that ATP (adenosine triphosphate), a molecule well known to biochemists for its role as a source of energy inside cells, could also transmit signals between them. Initially, his somewhat heretical theory, that did not fit conventional views, found considerable resistance in the scientific community. However, he continued to accumulate evidence in favor of his hypothesis, extending it to a variety of organs and systems and demonstrating a role for purinergic signaling in the cardiovascular, respiratory and nervous systems, and in the pathophysiology of pain, blood clotting, cell proliferation and differentiation, and immunity. For his entire life, he struggled to attract scientists to this new field and, finally, in the early 1990s, did evidence emerge that convinced the doubters, due to new molecular biology techniques making it possible to isolate and identify the cell surface receptors for ATP and its breakdown product, adenosine. His death clearly impacted a huge number of scientists who have lost their pioneering leader. In this Review, I will not talk of the many discoveries made by Professor Burnstock, nor of his enormous scientific contributions to the field and of the incredible number of prizes and public recognitions that he has received after his theory was accepted worldwide. Instead, I will share some personal memories on him as a teacher and scientist, and, most of all, as a loyal and reliable friend. Geoff was an extraordinary human being, always eager to collaborate and share data, never jealous of his findings and capable of learning even from young people. He was known for his enthusiasm, empathy and ability to motivate young scientists. I was lucky to meet him when I was still very young, and the collaboration and friendship that we established and maintained across the years has profoundly conditioned my professional and personal life. For me, Geoff was what in Italy we call a "Maestro", one of those leading figures who are fundamental not only for mentoring an individual's career but also their growth as a scientist and as a human being.

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

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.

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

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 SOD1G93A 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 SOD1G93A 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.

Dipeptidyl peptidase-4 inhibitors and sulfonylureas prevent the progressive impairment of the nigrostriatal dopaminergic system induced by diabetes during aging.

The nigrostriatal dopaminergic system (NDS) controls motor activity, and its impairment during type 2 diabetes (T2D) progression could increase Parkinson's disease risk in diabetics. If so, whether glycemia regulation prevents this impairment needs to be addressed. We investigated whether T2D impairs the NDS and whether dipeptidyl peptidase-4 inhibition (DPP-4i; a clinical strategy against T2D but also neuroprotective in animal models) prevents this effect, in middle-aged mice. Neither T2D (induced by 12 months of high-fat diet) nor aging (14 months) changed striatal dopamine content assessed by high-performance liquid chromatography. However, T2D reduced basal and amphetamine-stimulated striatal extracellular dopamine, assessed by microdialysis. Both the DPP-4i linagliptin and the sulfonylurea glimepiride (an antidiabetic comparator unrelated to DPP-4i) counteracted these effects. The functional T2D-induced effects did not correlate with NDS neuronal/glial alterations. However, aging itself affected striatal neurons/glia, and the glia effects were counteracted mainly by DPP-4i. These findings show NDS functional pathophysiology in T2D and suggest the preventive use of two unrelated anti-T2D drugs. Moreover, DPP-4i counteracted striatal age-related glial alterations suggesting striatal rejuvenation properties.

In vivo silencing of miR-125a-3p promotes myelin repair in models of white matter demyelination.

In the last decade, microRNAs have been increasingly recognized as key modulators of glial development. Recently, we identified miR-125a-3p as a new player in oligodendrocyte physiology, regulating in vitro differentiation of oligodendrocyte precursor cells (OPCs). Here, we show that miR-125a-3p is upregulated in active lesions of multiple sclerosis (MS) patients and in OPCs isolated from the spinal cord of chronic experimental autoimmune encephalomyelitis (EAE) mice, but not in those isolated from the spontaneously remyelinating corpus callosum of lysolecithin-treated mice. To test whether a sustained expression of miR-125a-3p in OPCs contribute to defective remyelination, we modulated miR-125a-3p expression in vivo and ex vivo after lysolecithin-induced demyelination. We found that lentiviral over-expression of miR-125a-3p impaired OPC maturation, whereas its downregulation accelerated remyelination. Transcriptome analysis and luciferase reporter assay revealed that these effects are partly mediated by the direct interaction of miR-125a-3p with Slc8a3, a sodium-calcium membrane transporter, and identified novel candidate targets, such as Gas7, that we demonstrated necessary to correctly address oligodendrocytes to terminal maturation. These findings show that miR-125a-3p upregulation negatively affects OPC maturation in vivo, suggest its role in the pathogenesis of demyelinating diseases and unveil new targets for future promyelinating protective interventions.

Regulation and signaling of the GPR17 receptor in oligodendroglial cells.

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.

Adenosine Signaling in Glioma Cells.

Purines 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 tumours (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 tumours. 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. Additionally, we have included new original data from our laboratory demonstrating a key involvement of MAP kinases in the cytostastic and cytotoxic effects exerted by an adenosine analogue, 2-CdA, which with the name of Cladribine is already clinically utilized in haematological malignancies. Here we show that 2-CdA can activate multiple intracellular pathways leading to cell cycle block and cell death by apoptosis of a human astrocytoma cell line that bears several pro-survival genetic mutations. Although in vivo data are still lacking, our results suggest that adenosine analogues could therefore be exploited to overcome resistance to chemotherapy of brain tumours.

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

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.