Pharmacological and biotechnological in vitro approaches unveil the role of GPR17 signaling in regulating the timing of oligodendroglial differentiation

Background and Purpose - In the adult central nervous system there are many oligodendrocyte precursor cells (OPCs) that serve as the primary source of remyelinating cells in demyelinated lesions. Knowledge of the mechanisms regulating OPC maturation is needed to unveil novel pharmacological targets in demyelinating diseases. The G-protein-coupled membrane receptor GPR17, activated by both uracil nucleotides and cysteinyl-leucotrienes [1], has recently emerged as an important player in oligodendrogliogenesis [2,3]. It has been previously reported that GPR17 presence is restricted to NG2+-OPCs at early differentiation stages and is completely segregated from that of myelin proteins [4]. Here, we used purified primary OPCs from rat cortical parenchyma to assess the functional consequences of GPR17 modulation by either pharmacological or biotechnological approaches on the differentiation program of these cells. Methods and Results - OPCs were exposed to the GPR17 agonists UDP-glucose, UDP and LTE4 for 48 hours. The degree of OPC differentiation was assessed on fixed cultures by immunostaining with an antibody against Myelin basic protein (MBP), a marker of mature oligodendrocytes. Data show that all these agonists increase the proportion of MBP+ cells compared to controls, suggesting acceleration of cell maturation by promoting receptor activation. Secondly, transfection experiments with fluorescent plasmids, enabling either silencing or over-expression of GPR17 were performed to univocally correlate the expression of this receptor with cell shape changes and phenotype acquisition during oligodendroglial maturation. Preliminary results show that suppression of GPR17 expression at early differentiation stages reduces the number of MBP+cells in culture, indicating that its silencing impairs the normal program of OPC differentiation. Conclusions - Globally, these data point at GPR17 as a key regulator of oligodendrogliogenesis and at GPR17 ligands as extrinsic local regulators of OPCs under physiological conditions and during myelin repair. References [1] P. Ciana, M. Fumagalli, M.L. Trincavelli, C. Verderio, P. Rosa, D. Lecca, S. Ferrario, C. Parravicini, V. Capra, P. Gelosa, U. Guerrini, S. Belcredito, M. Cimino, L. Sironi, E. Tremoli, G.E. Rovati, C. Martini, M.P. Abbracchio, The orphan receptor GPR17 identified as a new dual uracil nucleotides/cysteinil-leukotrienes receptor. EMBO J, 19, 4615-2627, 2006. [2] D. Lecca, M.L. Trincavelli, P. Gelosa, L. Sironi, P. Ciana, M. Fumagalli, G. Villa, C. Verderio, C. Grumelli, U. Guerrini, E. Tremoli, P. Rosa, S. Cuboni, C. Martini, A. Buffo, M Cimino, M.P. Abbracchio, The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair. PloS One, 10, e3579, 2008. [3] Y. Chen, H. Wu, S. Wang, H. Koito, J. Li, F. Ye, J. Hoang, S.S. Escobar, A. Gow, H.A. Arnett, B.D. Trapp, N.J. Karandikar, J. Hsieh, Q.R. Lu, The oligodendrocyte-specific G protein-coupled receptor GPR17 is a cell-intrinsic timer of myelination. Nature Neuroscience, 12, 1398-1406. [4] M. Fumagalli, S. Daniele, D. Lecca, P.R. Lee, C. Parravicini, R.D. Fields,P. Rosa, F. Antonucci, C. Verderio, M.L Trincavelli, P. Bramanti, C. Martini, M.P. Abbracchio, Phenotypic changes, signaling pathway, and functional correlates of GPR17-expressing neural precursor cells during oligodendrocyte differentiation. The Journal of biology chemistry,12, 10593-10604.