Biotechnological and pharmacological in vitro approaches unveil the role of GPR17 signaling in regulating the timing of oligodendroglial differentiation

The G-protein-coupled membrane receptor GPR17, activated by both uracil nucleotides and cysteinyl-leucotrienes (Ciana et al., 2006), has recently emerged as an important player in oligodendrogliogenesis (Lecca et al., 2008; Chen et al., 2009). Here, we used purified oligodendrocyte precursor cells (OPCs) to fully define the immunophenotype of the GPR17-expressing cells during OPC differentiation, unveil its native signaling pathway and assess the functional consequences of GPR17 modulation by either biotechnological and pharmacological approaches on the differentiation program of these cells. Real-time PCR and immunocytochemical experiments showed distinct GPR17 mRNA and protein levels of expression that define different steps of OPCs maturation, ranging from the precursor stage to the pre-myelinating phase. Specifically, at early stages, a fraction of slowly proliferating and morphologically-immature OPCs, expressing the proteoglycan NG2 and other early proteins like Olig2 and PDGF receptor-α, displayed GPR17 labeling. Receptor expression gradually increased with in vitro morphological differentiation in cells with more processes emerging from the cell body, was maximal in immature pre-oligodendrocytes expressing the markers O4 and O1, whereas was turned down in morphologically mature cells positive for the myelin proteins PLP and MBP. Thus, GPR17 presence was restricted to very early differentiation stages and completely segregated from that of mature myelin. In OPCs, GPR17 is functional and its activation, by either uracil nucleotides or cysLTs, resulted in potent inhibition of intracellular cAMP formation. This effect was counteracted by GPR17 antagonists and receptor silencing with siRNAs. Inhibition of adenyl cyclase thus represents the primary transduction signaling system of the native receptor. Of note, uracil nucleotides promoted and GPR17 inhibition (by either antagonists or siRNAs) impaired the normal program of OPC differentiation, indicating that GPR17 signaling regulates the timing of oligodendroglial differentiation. These data may have implications for the in vivo behaviour of adult OPCs and point to uracil nucleotides and cysLTs as main extrinsic local regulators of these cells under physiological conditions and during myelin repair (Fumagalli et al., 2011). Ciana et al. (2005) Embo J. 25, 4615-4627. Lecca et al. (2008) PLoS One. 3, e3579. Chen et al. (2009) Nat Neurosci. 12, 1398-1406. Fumagalli et al. (2011) J Biol Chem 286, 10593-604.