Adenosine A2B receptors stimulation inhibits oligodendrocyte maturation by interacting with sphingosine kinase/sphingosine 1-phosphate signalling axis in primary purified oligodendrocyte cultures

Multiple Sclerosis (MS) is the most frequent demyelinating disease in the Central Nervous System (CNS). Remyelination does occur, but is limited especially in chronic disease stages. Despite effective immunomodulatory therapies reduce the number of relapses, the progressive disease phase cannot be prevented. Therefore, promotion of neuroprotective and repair mechanisms, such as remyelination, represents an attractive additional treatment strategy. A number of pathways have been identified that may contribute to ameliorate the impaired remyelination in MS lesions; among them adenosinergic signalling and sphingosine kinase/sphingosine 1-phosphate signalling axis (SphK/S1P). Oligodendrocyte development involves progression from oligodendrocyte progenitor cells (OPCs) to mature oligodendrocytes. Oligodendrocytes express each of the different adenosine receptor subtypes (A1, A2A, A2B and A3) at all maturational stage. Interestingly, treatment ofOPCs in culture with adenosine promotesmaturation, an effect that is A1 receptor mediated [1]. Activation of A1 receptors also stimulated OPC migration, without adverse effects on cell viability [2]. Treatment of OPCs in culture with selective A2A agonists reduces cellmaturation [3,4]. Avery recent study demonstrated that Fingolimod (FTY720), that has been approved as orally active drug for relapsing MS, modulates S1P receptors. A relationship between SphK1 activity and A2B adenosine receptor (ADORA2B) activation has been demonstrated in mouse normal and sickle erythrocytes in vitro [5]. The interaction between ADORA2B and S1P/SphK signalling on oligodendrocyte maturation in rat cultured OPCs, at different times of maturation, was investigated performing Real-time PCR and Western Blot analysis. In cultured OPCs, SphK1 phosphorylation, that is a hallmark of the activation state of the enzyme, was enhanced after a 10 min treatment with BAY60- 6583 (10 μM), a selective ADORA2B agonist, thus demonstrating an interaction between ADORA2B activation and SphK1. Chronic application (7 days) of BAY60-6583 (1-10 μM) or of the newly synthetized ADORA2B agonist, P453 (50-100 nM) in cultured medium reduced OPC differentiation, as indicated by the decrease of the two genes target MAG (myelin-associated glycoprotein) and Mbp3 (myelin basic protein 3), typically expressed by mature oligodendrocytes. FTY720 phosphate (1 μM), the active metabolite of FTY720, mimicked the effect of 10 μM BAY60-6583 on OPCmaturation. On the contrary, VPC96047 (0.5 μM), a pan-SphK inhibitor, and VPC96091 (0.5 μM) a selective SphK1 inhibitor, increasedMAG and Mbp3 levels. These effects were abolished in the presence of 10 μM BAY60-6583. Our findings reveal a novel signalling network i.e. ADORA2B and SphK1 regulating remyelination process in rat cultured OPCs.