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An aberrant pro-inflammatory microglia response has been associated with most neurodegenerative disorders. Identifying microglia druggable checkpoints to restore their physiological functions is an emerging challenge. Recent data have shown that microglia produce de novo neurosteroids, endogenous molecules exerting potent anti-inflammatory activity. Here, the role of neurosteroidogenesis in the modulation of microgliosis was explored in human microglia cells. In particular, CYP11A1 inhibition or TSPO pharmacological stimulation, crucial proteins involved in the rate limiting step of the neurosteroidogenic cascade, were employed. CYP11A1 inhibition led microglia to acquire a dysfunctional and hyperreactive phenotype, while selective TSPO ligands promoted the establishment of an anti-inflammatory one. Analysis of specific neurosteroid levels (neurosteroidome) identified allopregnanolone/pregnanolone as crucial metabolites allowing controlled activation of microglia. Importantly, the neurosteroid shift towards a greater androgenic/estrogenic profile supported the transition from pro-inflammatory to neuroprotective microglia, suggesting the therapeutic potential of de novo microglial neurosteroidogenesis stimulation for neuroinflammatory-related disorders. [ABSTRACT FROM AUTHOR]

Glioblastoma (GB) is a lethal brain tumor that rapidly adapts to the dynamic changes of the tumor microenvironment (TME). Mesenchymal stem/stromal cells (MSCs) are one of the stromal components of the TME playing multiple roles in tumor progression. GB progression is prompted by the immunosuppressive microenvironment characterized by high concentrations of the nucleoside adenosine (ADO). ADO acts as a signaling molecule through adenosine receptors (ARs) but also as a genetic and metabolic regulator. Herein, the effects of high extracellular ADO concentrations were investigated in a human glioblastoma cellular model (U343MG) and MSCs. The modulation of the purinome machinery, i.e., the ADO production (CD39, CD73, and adenosine kinase [ADK]), transport (equilibrative nucleoside transporters 1 (ENT1) and 2 (ENT2)), and degradation (adenosine deaminase [ADA]) were investigated in both cell lines to evaluate if ADO could affect its cell management in a positive or negative feed‐back loop. Results evidenced a different behavior of GB and MSC cells upon exposure to high extracellular ADO levels: U343MG were less sensitive to the ADO concentration and only a slight increase in ADK and ENT1 was evidenced. Conversely, in MSCs, the high extracellular ADO levels reduced the ADK, ENT1, and ENT2 expression, which further sustained the increase of extracellular ADO. Of note, MSCs primed with the GB‐conditioned medium or co‐cultured with U343MG cells were not affected by the increase of extracellular ADO. These results evidenced how long exposure to ADO could produce different effects on cancer cells with respect to MSCs, revealing a negative feedback loop that can support the GB immunosuppressive microenvironment. These results improve the knowledge of the ADO role in the maintenance of TME, which should be considered in the development of therapeutic strategies targeting adenosine pathways as well as cell‐based strategies using MSCs. [ABSTRACT FROM AUTHOR]

GPR17, a G protein-coupled receptor, is a pivotal regulator of myelination. Its endogenous ligands trigger receptor desensitization and downregulation allowing oligodendrocyte terminal maturation. In addition to its endogenous agonists, GPR17 could be promiscuously activated by pro-inflammatory oxysterols and chemokines released at demyelinating lesions. Herein, the chemokine receptors CXCR2 and CXCR4 were selected to perform both in silico modelling and in vitro experiments to establish their structural and functional interactions with GPR17. The relative propensity of GPR17 and CXCR2 or CXCR4 to form homo- and hetero-dimers was assessed by homology modelling and molecular dynamics (MD) simulations, and co-immunoprecipitation and immunoenzymatic assay. The interaction between chemokine receptors and GPR17 was investigated by determining receptor-mediated modulation of intracellular cyclic adenosine monophosphate (cAMP). Our data show the GPR17 association with CXCR2 or CXCR4 and the negative regulation of these interactions by CXCR agonists or antagonists. Moreover, GPR17 and CXCR2 heterodimers can functionally influence each other. In contrast, CXCR4 can influence GPR17 functionality, but not vice versa. According to MD simulations, all the dimers reached conformational stability and negative formation energy, confirming the experimental observations. The cross-talk between these receptors could play a role in the development of the neuroinflammatory milieu associated with demyelinating events. [ABSTRACT FROM AUTHOR]

The previously 'orphan' G protein-coupled receptor GPR17 is structurally related to both P2Y nucleotide receptors and to receptors for cysteinyl leukotrienes. Genomic analysis revealed two putative open reading frames encoding for a 'short' and a 'long' receptor isoform of 339- and 367-amino acids, respectively, with the latter displaying a 28-amino acid longer N[H.sub.2] terminus. The short isoform has been recently 'deorphanized,' revealing dual responses to uracil nucleotides and cysteinyl leukotrienes. No information regarding the ligand specificity, tissue distribution, or pathophysiological roles of the long receptor isoform is available. In the present study, we cloned human long-GPR17, determined its tissue distribution, and characterized its pharmacological specificity in 1321N1 cells by [[sup.35]S]GTP[gamma]S binding (which measures the ability of G protein-coupled receptor agonists to increase GTP binding to G proteins) and whole cell patch-clamp recording measuring receptor coupling to [K.sup.+] channels. [[sup.35]S]GTP[gamma]/S binding in long-GPR 17-expressing 1321N 1 cells revealed concentration-dependent responses to uracil nucleotides (UDP-galactose = UDP > UDP-glucose) and cysteinyl leukotrienes (LT[C.sub.4] > LT[D.sub.4]), which were counteracted by a purinergic (cangrelor) and a cysteinyl leukotriene antagonist (montelukast), respectively. The nonhydrolyzable ATP analog ATP[gamma]S also acted as an antagonist. GPR17 coupled to Gi and, to a lesser extent, [G.sub.q] proteins. UDP-glucose and LT[D.sub.4] also induced increases in overall outward [K.sup.+] currents, which were antagonized by the pufinergic antagonists MRS2179 and cangrelor and by montelukast. We conclude that the previously uncharacterized long-GPR17 isoform is a functional receptor that is stimulated by both uracil nucleotides and cysteinyl leukotrienes. We also show that the signaling pathway of GPR17 involves the generation of outward [K.sup.+] currents, an important protective mechanism that, in brain, is specifically aimed at reducing neuronal hyperexcitability and resultant neuronal injury. uracil nucleotides; cysteinyl leukotrienes; G protein-coupled receptors; big-conductance [K.sup.+] channels; neuronal damage; guanosine 5'-O-(3-thiotriphosphate) doi: 10.1152/ajpcell.00658.2008