Anales RANF

P.57 ADENOSINE A 2B RECEPTORS AND SPHINGOSINE KINASE/SPHINGOSINE 1-PHOSPHATE SIGNALING AXIS CONTROL MATURATION OF OLIGODENDROCYTE PRECURSOR CELLS IN VITRO E. Coppi, I. Fusco, F. Cherchi, I. Dettori, F. Cencetti, L. Gaviano, V. Colotta, D. Catarzi, P. Bruni, F. Pedata and A.M. Pugliese University of Florence, Florence, Italy. Introduction : Restoration of the myelin sheaths requires the differentiation of the oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs). Adenosine and sphingosine kinase/sphingosine1-phosphate signaling axis (SphK/S1P) play important roles in remyelination processes. Remarkably, Fingolimod (FTY720), approved as orally active drug for relapsing Multiple Sclerosis, also modulates S1P receptors. An interaction between adenosine A2B receptors (ADORAA2B) and SphK activity has been demonstrated in mouse and human normal and sickle erythrocyte (Sun et al., 2015, Blood 125(10):1643-52). The voltage-dependent K + current (I K ) block by tetraethylammonium (TEA) is also known to impair OPC maturation (Gallo et al., 1996, J Neurosci 16(8):2659-70). Material and methods : The role of ADORA2B and SphK/S1P signaling on oligodendrogenesis in rat cultured OPCs was investigated by patch clamp, Real Time PCR and Western Blot techniques. Results : The BAY60-6583 (0.1-30 µM), a selective ADORA2B agonist, reduced outward currents evoked by a voltage ramp protocol. This effect was blocked by MRS1706 (10 µM), a selective ADORA2B antagonist. The effect of BAY60-6583 on outward currents was prevented by TEA, indicating the involvement of I K in BAY60- 6583-mediated effect. The phosphorylated form of FTY720 (FTY720-P 1 µM) mimicked and partially occluded the effect of 10 µM BAY60-6583. SphK1 phosphorylation was enhanced after acute treatment with BAY60-6583, demonstrating an interaction between SphK/S1P pathway and ADORA2B activation. Prolonged ADORA2B stimulation reduced the expression of mature OL markers in cultured OPC. In contrast, SphK inhibitors produced an opposite effect. Finally, we found that downregulation of ADORA2B by siRNA significantly reduced the levels of NG2, S1P 5 receptors and SphK1 mRNAs and increased S1Plyase, suggesting that ADORA2B silencing stimulates OPC differentiation, modulates S1P-related target genes and reduces S1P levels. Conclusions : Our data shows that ADORA2B stimulation inhibits K+ channels necessary to OPC differentiation and interferes with SphK/S1P pathway. Furthermore, either ADORA2B stimulation and S1P production inhibit OPC maturation.