High adenosine extracellular levels induce glioblastoma aggressive traits modulating the mesenchymal stromal cell secretome

Glioblastoma is an aggressive, fast-growing brain tumor influenced by the composition of the tumor microenvironment (TME) in which mesenchymal stromal cell (MSCs) play a pivotal role. Adenosine (ADO), a purinergic signal molecule, can reach up to high micromolar concentrations in TME. The activity of specific adenosine receptor subtypes on glioma cells has been widely explored, as have the effects of MSCs on tumor progression. However, the effects of high levels of ADO on glioma aggressive traits are still unclear as is its role in cancer cells-MSC cross-talk. Herein, we first studied the role of extracellular Adenosine (ADO) on isolated human U343MG cells as a glioblastoma cellular model, finding that at high concentrations it was able to prompt the gene expression of Snail and ZEB1, which regulate the epithelial–mesenchymal transition (EMT) process, even if a complete transition was not reached. These effects were mediated by the induction of ERK1/2 phosphorylation. Additionally, ADO affected isolated bone marrow derived MSCs (BM-MSCs) by modifying the pattern of secreted inflammatory cytokines. Then, the conditioned medium (CM) of BM-MSCs stimulated with ADO and a co-culture system were used to investigate the role of extracellular ADO in GBM–MSC cross-talk. The CM promoted the increase of glioma motility and induced a partial phenotypic change of glioblastoma cells. These effects were maintained when U343MG cells and BM-MSCs were co-cultured. In conclusion, ADO may affect glioma biology directly and through the modulation of the paracrine factors released by MSCs overall promoting a more aggressive phenotype. These results point out the importance to deeply investigate the role of extracellular soluble factors in the glioma cross-talk with other cell types of the TME to better understand its pathological mechanisms

Synthesis and AT1 affinity evaluation of benzamidophenyl analogs of known AT1 receptor ligands with similar aromatic skeleton

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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

Phytochemical analysis and antisenescence activity of Sorbus torminalis (L.) Crantz and Elaeagnus umbellata Thunb fruits

In Italy, many spontaneous plants are used as food in folk traditions and are now being re-evaluated as healthy products with high nutritional value. In the ethnobotanical field, we selected a fruit tree that modern gastronomy has forgotten: the "Ciavardello" (Sorbus torminalis (L.) Crantz). The Italian phyto-alimentary tradition (1) uses its fruits to make jams, jellies, syrups, fresh snacks and, less often, alcoholic beverages. The "Ciavardello" is a species of Sorbus native to Europe, North Africa, and Asia Minor and it is a member of the Rosaceae family. It is a deciduous tree or shrub which grows 1-7 m tall (sometimes reaching 20 m) and it is a slow-growing and long-lived species found in forests of broadleaf trees. The trunk is straight and the crown is expanded, globose and dense, while the bark is smooth and greyish. The leaves are alternate, simple, glabrous, petiole, ovate (4–10 cm long and 2–8 cm wide), with five to nine acute lobes, serrate and dark green colored on both sides. The flowers are hermaphrodite, 5-merous, with white petals and they are produced in corymbs. It blooms in spring (April-May) and bears fruit in autumn (September-October). The fruit is a globose to ovoid pome 10–15 mm in diameter and it is greenish to russet or brown and patterned with small and pale lenticel spots when ripe, with a pleasantly acidulous taste (2). Another interesting plant is ‘Albero dei coralli’ (Elaeagnus umbellata Thunb.), an allochthonous species belonging to the Elaeagnaceae family. It is cultivated in Italy for ornamental purposes, while the fruits are eaten fresh, a custom that was imported from tropical and temperate Asia, its native region. The "Albero dei coralli" is a deciduous shrub or tree, more or less spiny, which grows 3-5 m tall. The leaves are alternate, lanceolate (4-10 cm x 2-4 cm), with wavy margins, green colored above and covered with silvery scales below. The flowers are hermaphrodite, fragrant, whitish, tubular and 4-lobed and they are found in the leaf axils in clusters of 1-7 elements. The fruits are small roundish drupes (3-9 mm diameter), reddish to pink, dotted with scales and they are pulpy, juicy and sweet. It blooms in the spring and its fruits ripen in the fall (2). Both fresh fruits, collected in the Tuscany region (Italy), were extracted at room temperature with EtOH-H2O 80% (three times, every 24 h) and obtained residues were partitioned between n-BuOH and H2O. The n-butanolic extracts were finally analyzed by HPLC-PDA/UV-ESI-MS/MS techniques. The chemical profile of S. torminalis revealed the presence of phenolic acids and flavonol glycosides (3), while E. umbellata extract was rich especially in quercetin and kaempferol derivatives. In the scenario of regenerative medicine, the gingival mesenchymal stem cells (GMSCs) have arisen as a promising tool to repair damaged tissues. Herein, the GMSCs were used to investigate the beneficial effects of n-butanolic extract of investigated fruits. Both extracts were able to increase the GMSC proliferation and decrease the intracellular accumulation of ROS. Furthermore, the extracts were able to counteract the senescence phenomena in GMSC with different extent. In particular, they contrast the ROS production mediated by hydroxyurea and hydrogen peroxide and reduced the age-related phenotypic changes (SA-β-gal staining). In conclusion, these results highlight S. torminalis and E. umbellata fruit extracts as novel sources of antioxidant phytocomplexes able to decrease the senescence process in mesenchymal cells. The ability of both extracts per se to ameliorate the GMSC well-being and decrease cellular senescence shed light on their possible use in regenerative medicine and in particular in all the GMSC in vitro application

High Levels of β-Amyloid, Tau, and Phospho-Tau in Red Blood Cells as Biomarkers of Neuropathology in Senescence-Accelerated Mouse

Alzheimer’s Disease (AD) is the most common Neurodegenerative Disease (ND), primarily characterised by neuroinflammation, neuronal plaques of β-amyloid (Aβ), and neurofibrillary tangles of hyperphosphorylated tau. α-Synuclein (α-syn) and its heteroaggregates with Aβ and tau have been recently included among the neuropathological elements of NDs. These pathological traits are not restricted to the brain, but they reach peripheral fluids as well. In this sense, Red Blood Cells (RBCs) are emerging as a good model to investigate the biochemical alterations of aging and NDs. Herein, the levels of homo- and heteroaggregates of ND-related proteins were analysed at different stages of disease progression. In particular, a validated animal model of AD, the SAMP8 (Senescence-Accelerated Mouse-Prone) and its control strain SAMR1 (Senescence-Accelerated Mouse-Resistant) were used in parallel experiments. The levels of the aforementioned proteins and of the inflammatory marker interleukin-1β (IL-1β) were examined in both brain and RBCs of SAMP8 and SAMR1 at 6 and 8 months. Brain Aβ, tau, and phospho-tau (p-tau) were higher in SAMP8 mice than in control mice and increased with AD progression. Similar accumulation kinetics were found in RBCs, even if slower. By contrast, α-syn and its heterocomplexes (α-syn-Aβ and α-syn-tau) displayed different accumulation kinetics between brain tissue and RBCs. Both brain and peripheral IL-1β levels were higher in SAMP8 mice, but increased sooner in RBCs, suggesting that inflammation might initiate at a peripheral level before affecting the brain. In conclusion, these results confirm RBCs as a valuable model for monitoring neurodegeneration, suggesting peripheral Aβ, tau, and p-tau as potential early biomarkers of AD

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

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

Co-localization and functional cross-talk between A1 and P2Y1 purine receptors in rat hippocampus

Adenosine and ATP, via their specific P1 and P2 receptors, modulate a wide variety of cellular and tissue functions, playing a neuroprotective or neurodegenerative role in brain damage conditions. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, recent data suggest the existence of a heterodimerization and a functional interaction between P1 and P2 receptors in the brain. In particular, interactions of adenosine A1 and P2Y1 receptors may play important roles in the purinergic signalling cascade. In the present work, we investigated the subcellular localization/co-localization of the receptors and their functional cross-talk at the membrane level in Wistar rat hippocampus. This is a particularly vulnerable brain area, which is sensitive to adenosine- and ATP-mediated control of glutamatergic transmission. The postembedding immunogold electron microscopy technique showed that the two receptors are co-localized at the synaptic membranes and surrounding astroglial membranes of glutamatergic synapses. To investigate the functional cross-talk between the two types of purinergic receptors, we evaluated the reciprocal effects of their activation on their G protein coupling. P2Y1 receptor stimulation impaired the potency of A1 receptor coupling to G protein, whereas the stimulation of A1 receptors increased the functional responsiveness of P2Y1 receptors. The results demonstrated an A1–P2Y1 receptor co-localization at glutamatergic synapses and surrounding astrocytes and a functional interaction between these receptors in hippocampus, suggesting ATP and adenosine can interact in purine-mediated signalling. This may be particularly important during pathological conditions, when large amounts of these mediators are released

The Recently Identified P2Y-Like Receptor GPR17 Is a Sensor of Brain Damage and a New Target for Brain Repair

Deciphering the mechanisms regulating the generation of new neurons and new oligodendrocytes, the myelinating cells of the central nervous system, is of paramount importance to address new strategies to replace endogenous damaged cells in the adult brain and foster repair in neurodegenerative diseases. Upon brain injury, the extracellular concentrations of nucleotides and cysteinyl-leukotrienes (cysLTs), two families of endogenous signaling molecules, are markedly increased at the site of damage, suggesting that they may act as “danger signals” to alert responses to tissue damage and start repair. Here we show that, in brain telencephalon, GPR17, a recently deorphanized receptor for both uracil nucleotides and cysLTs (e.g., UDP-glucose and LTD4), is normally present on neurons and on a subset of parenchymal quiescent oligodendrocyte precursor cells. We also show that induction of brain injury using an established focal ischemia model in the rodent induces profound spatiotemporal-dependent changes of GPR17. In the lesioned area, we observed an early and transient up-regulation of GPR17 in neurons expressing the cellular stress marker heat shock protein 70. Magnetic Resonance Imaging in living mice showed that the in vivo pharmacological or biotechnological knock down of GPR17 markedly prevents brain infarct evolution, suggesting GPR17 as a mediator of neuronal death at this early ischemic stage. At later times after ischemia, GPR17 immuno-labeling appeared on microglia/macrophages infiltrating the lesioned area to indicate that GPR17 may also acts as a player in the remodeling of brain circuitries by microglia. At this later stage, parenchymal GPR17+ oligodendrocyte progenitors started proliferating in the peri-injured area, suggesting initiation of remyelination. To confirm a specific role for GPR17 in oligodendrocyte differentiation, the in vitro exposure of cortical pre-oligodendrocytes to the GPR17 endogenous ligands UDP-glucose and LTD4 promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a “sensor” that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis