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The current PhD thesis focuses on the role of sphingosine-1-phophate receptors (S1PRs) and their modulators in mice central nervous system (CNS). Beside the well-recognized immunomodulatory effect of the compounds, new insights are now suggesting that these molecules may directly interact with S1PRs located on CNS cells. In this context, the project aims at i) investigating the presence and the activity of S1PRs in mouse cortical synaptosomes (isolated nerve endings) and gliosomes (astrocytic processes) in both control and EAE mice ii) studying the effect of siponimod (BAF312) on oligodendroglia cells differentiation. While the first part of the project was carried out at the University of Genoa (Italy), experiments on oligodendroglial cells were performed at the University of Hasselt (Belgium). Firstly, we performed Western blot and confocal analyses which confirmed the existence of S1P1R and S1P3R in both glutamatergic cortical synaptosomes and gliosomes. Then, we performed functional studies (superfusion experiments) to investigate the effect of S1PRs activation in controlling glutamate transmission. By using different ligands (the broad-spectrum agonist sphingosine-1-phosphate (S1P), the S1P1R agonist CS-2100 and the selective S1P3R agonist CYM-5541) we demonstrated that presynaptic S1P1Rs are able to inhibit the 12 mM KCl-evoked release of preloaded [3H]D-aspartate (an analogue of glutamate) in cortical synaptosomes, while the activation of presynaptic S1P3Rs significantly enhances it. In contrast, the S1P1R agonist was ineffective in cortical gliosomes while CYM-5541 inhibited the 15 mM KCl-evoked [3H]D-aspartate exocytosis from cortical gliosomes. The opposite outcomes can be explained by the coupling of the receptors to different types of G-protein which, when activated, can generate opposite intracellular signals. The hypothesis is further support by the evidence that S1P failed to affect the [3H]D-aspartate release in both cortical preparations. The concomitant activation of both S1P1R and S1P3R avoid the possibility to observe a clear effect in glutamate transmission in presence of this agonist. Then, we investigated the expression of both receptors in cortical synaptosomes and gliosomes from experimental autoimmune encephalomyelitis (EAE). In particular, S1P1R and S1P3R are significantly increased in cortical synaptosomal lysates from EAE mice at the acute stage of disease, but unchanged in the gliosomal preparation. Interestingly, oral therapeutic administration of fingolimod (FTY720; 0.03 mg/Kg/day for 14 days) recovered the receptor densities to almost physiological levels and also restored their release-regulating activity. Surprisingly, biotinylation studies performed in synaptosomes and gliosomes from healthy mice suggested that the receptors are not expressed in the outer layer on the cellular membrane, since the S1P1R and S1P3R immunopositivity was absent in the biotinylated samples from both preparations. This evidence suggests that S1P1R and S1P3R may be preferentially located intracellularly or in the inner side of the plasma membrane. Similarly, biotinylation studies performed on control and EAE mice unveiled no changes in the membrane expression of the receptors.The results obtained in this first part improve the knowledge on the existence and the release-regulating activity of S1PRs in components of the tripartite synapsis (namely the presynaptic and the astrocytic one). All experiments were performed on cortical samples and not on the spinal cord, where inflammatory infiltration is known to be much higher in EAE animal model. The choice of this specific area for our investigation allowed to study the effects of FTY720 beyond its immunomodulatory activity, adding new insights on the effect of this S1PRs modulator on glutamatergic transmission. The second part of the project focused on the effect of another S1PRs modulator (siponimod, BAF312) on OPC in vitro differentiation. Despite very preliminary, the results suggested that treatment with BAF312 (10 nM) seems to induce a partial OPC differentiation as we can observed by the outcomes emerged from the immunostaining images as well as by the qPCR analysis. In parallel, at the same concentration, the treatment with BAF312 seems to enhance both TrkB mRNA expression and p-TrkB protein density. As an increased release of neurotrophic factors has been observed in literature following FTY720 administration, the increased expression and activation of TrkB receptors could represent an indirect way in which the drug can exert its neuroprotective effect. Further experiments will be conducted to confirm the preliminary results and study the effective role of BAF312 in these cells.

Multiple sclerosis (MS) is an autoimmune inflammatory disease characterized by demyelination, axonal loss, and synaptic impairment in the central nervous system (CNS). The available therapies aim to reduce the severity of the pathology during the early inflammatory stages, but they are not effective in the chronic stage of the disease. In this phase, failure in endogenous remyelination is associated with the impairment of oligodendrocytes progenitor cells (OPCs) to migrate and differentiate into mature myelinating oligodendrocytes. Therefore, stimulating differentiation of OPCs into myelinating oligodendrocytes has become one of the main goals of new therapeutic approaches for MS. Different disease-modifying therapies targeting sphingosine-1-phosphate receptors (S1PRs) have been approved or are being developed to treat MS. Besides their immunomodulatory effects, growing evidence suggests that targeting S1PRs modulates mechanisms beyond immunomodulation, such as remyelination. In this context, this review focuses on the current understanding of S1PR modulators and their direct effect on OPCs and oligodendrocytes. This work was funded by "stichting charcot foundation" and "FWO". Vanmierlo, T (corresponding author), Hasselt Univ, Biomed Res Inst, Neuroimmune Connect & Repair Lab, B-3500 Hasselt, Belgium ; Maastricht Univ, Sch Mental Hlth & Neurosci, European Grad Sch Neurosci, Dept Psychiat & Neuropsychol, NL-6200 MD Maastricht, Netherlands. roggeri@difar.unige.it; melissa.schepers@uhasselt.be; assia.tiane@uhasselt.be; ben.rombaut@uhasselt.be; lieve.vanveggel@uhasselt.be; niels.hellings@uhasselt.be; jos.prickaerts@maastrichtuniversity.nl; pittalug@difar.unige.it; tim.vanmierlo@uhasselt.be