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

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, Ca2+ release from radial glia mediated by P2Y1 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

Basal astrocyte and microglia activation in the central nervous system of Familial Hemiplegic Migraine Type I mice

Background Gain-of-function missense mutations in the alpha(1A) subunit of neuronal Ca(V)2.1 channels, which define Familial Hemiplegic Migraine Type 1 (FHM1), result in enhanced cortical glutamatergic transmission and a higher susceptibility to cortical spreading depolarization. It is now well established that neurons signal to surrounding glial cells, namely astrocytes and microglia, in the central nervous system, which in turn become activated and in pathological conditions can sustain neuroinflammation. We and others previously demonstrated an increased activation of pro-algogenic pathways, paralleled by augmented macrophage infiltration, in both isolated trigeminal ganglia and mixed trigeminal ganglion neuron-satellite glial cell cultures of FHM1 mutant mice. Hence, we hypothesize that astrocyte and microglia activation may occur in parallel in the central nervous system. Methods We have evaluated signs of reactive glia in brains from naive FHM1 mutant mice in comparison with wild type animals by immunohistochemistry and Western blotting. Results Here we show for the first time signs of reactive astrogliosis and microglia activation in the naive FHM1 mutant mouse brain. Conclusions Our data reinforce the involvement of glial cells in migraine, and suggest that modulating such activation may represent an innovative approach to reduce pathology

The Dual Behaviour of a GPCR Involved in Brain Damage an Repair: Forced Unbinding of the Receptor GPR17 Ligands from Wild Type and R255I Mutant Models Through a Computational Approach

GPR17 is a hybrid G-protein-coupled receptor activated by two unrelated ligand families, extracellular nucleotides and cysteinyl-leukotrienes, and involved in brain damage and repair. Its exploitment as a target for novel neuroreparative strategies depends on the elucidation of the molecular determinants driving binding of its ligands. We applied docking and molecular dynamics simulations to analyse the binding and the forced unbinding of two GPR17 ligands (the purinergic agonist UDP and the leukotriene receptor antagonist pranlukast) from both the wild-type receptor and a mutant model, where a basic residue hypothesized to be crucial for nucleotide binding had been mutated (R255I). Molecular dynamics suggested that GPR17 nucleotide binding pocket is enclosed between the helical bundle and EL2. The driving interaction involves R255 and the UDP phosphate moiety. Steered molecular dynamics experiments showed that the energy required to unbind UDP is higher for the wild-type receptor than for R255I. Three potential binding sites for pranlukast were found. In one of its preferential docking conformations, pranlukast tetrazole group is close to R255 and phenyl rings are placed into a subpocket highly conserved among GPCRs. Pulling forces developed to break polar and aromatic interactions of pranlukast were comparable. No differences between the wild-type receptor and the R255I receptor were found for the unbinding of pranlukast. These data suggest a crucial role for R255 in binding of nucleotides to GPR17. Aromatic interactions are instead likely to play a predominant role in the recognition of pranlukast, suggesting that two different binding subsites are present on GPR17

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

Lupin Peptide T9 (GQEQSHQDEGVIVR) Modulates the Mutant PCSK9D374Y Pathway : in vitro Characterization of its Dual Hypocholesterolemic Behavior

GQEQSHQDEGVIVR (T9) is a peptide originated by the tryptic digestion of lupin \u3b2-conglutin that is absorbed in human intestinal Caco-2 cells. A previous study has shown that T9 impairs the protein-protein interaction between mutant D374Y Proprotein Convertase Subtilisin/Kexin 9 (PCSK9D374Y) and the low-density lipoprotein receptor (LDLR), thus exerting a hypocholesterolemic effect. Moreover, a bioinformatic study predicting that T9 may potentially act as an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCoAR), has suggested a complementary cholesterol-lowering activity. The present study demonstrates that T9 inhibits in vitro the HMGCoAR functionality with an IC50 value of 99.5 \ub1 0.56 \ub5M. Through the inhibition of either HMGCoAR or PCSK9D374Y activities, T9 enhances the LDLR protein levels leading to an improved ability of HepG2 cells transfected with the mutant PCSK9D374Y-FLAG plasmid to uptake extracellular LDL with a final cholesterol-lowering effect. In addition, T9 modulates the PCSK9D374Y signaling pathway in transfected HepG2 cells leading to a decrease of PCSK9D374Y and HNF-1\u3b1 protein levels. All these results indicate that the hypocholesterolemic effects of T9 are due to a dual mechanism of action involving either the modulation of the PCSK9D374Y or LDLR pathways. This may represent an added value from a therapeutic point of view

Volterra equations perturbed by noise

We consider a linear abstract Volterra integrodifferential equation in a Hilbert space, forced by a Gaussian process. The equation involves a completely monotone convolution kernel with a singularity at t = 0 and a sectorial linear spatial operator. Existence and uniqueness of a weak solution is established. Furthermore we give conditions such that the solution converges to a stationary process. Our method consists in a state space setting so that the corresponding solution process is Markovian, and the tools of linear analytic semigroup theory can be utilized

Expression of GPR17 receptor in a murine model of perinatal brain neuroinflammation and its possible interaction with Wnt pathway

Oligodendrocyte precursor cells (OPCs) are generated in specific germinal regions and progressively maturate to myelinating cells. Oligodendrocytes (OLs) differentiation is regulated by a complex interplay of intrinsic, epigenetic and extrinsic factors, including Wnt and the G protein-coupled receptor referred to as GPR17 (Mitew et al., 2014). This receptor responds to both extracellular nucleotides (UDP, UDP-glucose) and cysteinyl-leukotrienes (Ciana et al., 2006), endogenous signaling molecules involved in inflammatory response and in the repair of brain lesions. GPR17 is highly expressed in OPCs during the transition to immature OLs, but it is down-regulated in mature cells. Accordingly, GPR17-expressing OPCs are already present in mice at birth, increase over time, reach a peak at P10, before the peak of myelination, and then decline in the adult brain (Boda et al., 2011). Of note, in cultured OPCs, early GPR17 silencing has been shown to profoundly affect their ability to generate mature OLs (Fumagalli et al., 2011, 2015). Myelination defects characterize many brain disorders, including perinatal brain injury caused by systemic inflammation (Favrais et al., 2011), which is a leading cause of preterm birth. It has already been suggested that an imbalance in the Wnt/\u3b2-catenin/TCF4 pathway could be involved in the maturation arrest of OLs that is observed in premature infants (Yuen et al., 2014). No data are currently available on GPR17 in perinatal brain injury and on its possible interaction with Wnt pathway. Based on these premises, the aim of this work was to assess if the maturational blockade of OLs due to mild systemic perinatal inflammation, induced by intraperitoneal injections of interleukin-1\u3b2 (IL- 1\u3b2), is accompanied by defects in GPR17 expression and whether the Wnt pathway is involved in the regulation of GPR17. Data showed that in newborn mice exposed to IL-1\u3b2, which induces a blockade of oligodendrocyte maturation, GPR17 expression is not affected at early time point (P5), but it is downregulated at P10, when its expression should be maximal. Moreover, in vitro studies revealed that the maturation blockade of the oligodendroglial cell line Oli-Neu, after treatment with a Wnt Agonist II, is accompanied by a severe inhibition of GPR17 expression. In conclusion, our data have shown that myelination defects observed in perinatal brain injury are associated with defects in GPR17 expression; further studies are needed to characterize the molecular link between Wnt pathway and GPR17 receptor

Gene regulation of GPR17, a checkpoint receptor in oligodendroglial differentiation

GPR17 is a G protein-coupled receptor activated by both uracil nucleotides and cysteinyl-leukotrienes. We have previously demonstrated GPR17 is a key regulator of oligodendroglial differentiation and myelination. The receptor starts to be expressed in early oligodendrocyte precursor cells (OPCs), it reaches its maximal expression in immature oligodendrocytes and is then progressively down-regulated. In late OPCs, GPR17 forced expression led to impaired maturation, suggesting that its expression needs to be tightly time regulated. Based on these evidences, this work was aimed at identifying the signaling molecules regulating GPR17 expression during oligodendroglial differentiation. For this purpose, we cloned a putative promoter region of Gpr17 into a reporter vector upstream to a gene encoding for a luciferase. Then, we transfected this construct in Oli-neu cells, an immortalized oligodendroglial cell line, and we set up a reporter assay to evaluate the bioluminescence produced in response to an array of stimuli. Our results showed that treatment with both dibutyryl-cAMP, an analogue of cAMP, and forskolin, an activator of adenylyl cyclase, led to a significant increase of promoter activity, suggesting that cAMP signaling triggers GPR17 expression. To evaluate if GPR17 could be regulated by neuronal factors, we incubated cells with medium conditioned by cortical neurons. After 48h, we observed a significant induction of promoter activity; this effect was enhanced by heating the medium, suggesting neurons release one or more factors promoting oligodendroglial differentiation via Gpr17 gene, but that an inhibitory thermolabile factor is also present in the neuronal-conditioned medium. In line with this hypothesis, we found that insulin, a component of the medium formulation known to activate the mTOR pathway, strongly inhibited GPR17 promoter activity, whereas rapamycin, an inhibitor of the same pathway, significantly increased it. These data are consistent with the hypothesis that, while a neuronal-derived product activating cAMP is involved in turning GPR17 on, the mTOR pathway, likely activated by insulin-like growth factors, may be responsible for its physiological silencing at later stages of oligodendroglial development. These results may be relevant to the identification of new pharmacological strategies to activate/inhibit GPR17 under dysregulated conditions accompanied by myelination defects

Mir-125a-3p negatively regulates oligodendrocyte precursor cells maturation and is altered in human multiple sclerosis

In the central nervous system, oligodendrocytes provide support to axons thanks to the production of a myelin sheath. During their maturation oligodendroglial precursors (OPCs) follow a very precise differentiation program, finely orchestrated by transcription factors, epigenetic factors and microRNAs, a class of small non-coding RNAs involved in post-transcriptional regulation. Any alterations in this program can potentially contribute to dysregulated myelination, impaired remyelination and neurodegenerative conditions, as it happens in multiple sclerosis. Recently, we identified miR-125a-3p as a new actor of oligodendroglial maturation, that could also be involved in the pathological consequences of multiple sclerosis, showing that its over-expression impairs, whereas its silencing promotes, oligodendrocyte maturation (Lecca et al., Sci Rep, 2016). To shed light on the mechanism underlying this effect, we performed a microarray analysis on OPCs after miR-125a-3p over-expression. This analysis suggested that miR-125a-3p is indeed involved in the regulation of biological processes important for OPC maturation, such as cell-cell interaction and morphological differentiation. To evaluate whether miR-125a-3p modulation may influence the progression of remyelination in vivo, we overexpressed the miR-125a-3p by lentiviral approach in a focal lysolecithin-mediated demyelinating lesion in the subcortical white matter of adult mice. Interestingly, also in this case, we found that miRNA-overexpressing OPCs persisted in an immature (i.e. PDGR\u3b1+/NG2+) state. Moreover, we found that miR-125a-3p levels are altered in both brain active lesions and cerebrospinal fluid of multiple sclerosis patients, suggesting that it could be a potential biomarker of disease. The identification of a new miRNA modulating oligodendrocyte differentiation provides new findings about the complex regulation of myelination processes and we postulate that an antago-miRNA for miR-125a-3p may help promoting oligodendrocyte maturation in diseases characterized by impaired myelin repair. Sponsored by Fondazione Italiana Sclerosi Multipla 2013/R-1 project to MPA and by Fondazione Cariplo, grant n\ub0 2014-1207 to DL

A new role for the P2Y-like GPR17 receptor in the modulation of multipotency of oligodendrocyte precursor cells in vitro

Oligodendrocyte precursor cells (OPCs, also called NG2 cells) are scattered throughout brain parenchyma, where they function as a reservoir to replace lost or damaged oligodendrocytes, the myelin-forming cells. The hypothesis that, under some circumstances, OPCs can actually behave as multipotent cells, thus generating astrocytes and neurons as well, has arisen from some in vitro and in vivo evidence, but the molecular pathways controlling this alternative fate of OPCs are not fully understood. Their identification would open new opportunities for neuronal replace strategies, by fostering the intrinsic ability of the brain to regenerate. Here, we show that the anti-epileptic epigenetic modulator valproic acid (VPA) can promote the generation of new neurons from NG2+ OPCs under neurogenic protocols in vitro, through their initial de-differentiation to a stem cell-like phenotype that then evolves to \u201chybrid\u201d cell population, showing OPC morphology but expressing the neuronal marker \u3b2III-tubulin and the GPR17 receptor, a key determinant in driving OPC transition towards myelinating oligodendrocytes. Under these conditions, the pharmacological blockade of the P2Y-like receptor GPR17 by cangrelor, a drug recently approved for human use, partially mimics the effects mediated by VPA thus accelerating cells\u2019 neurogenic conversion. These data show a co-localization between neuronal markers and GPR17 in vitro, and suggest that, besides its involvement in oligodendrogenesis, GPR17 can drive the fate of neural precursor cells by instructing precursors towards the neuronal lineage. Being a membrane receptor, GPR17 represents an ideal \u201cdruggable\u201d target to be exploited for innovative regenerative approaches to acute and chronic brain diseases