Biological role of sphingosine 1-phosphate in neuroblasts derived from otic vesicle

Hearing loss is a health and social problem, particularly common among older people. Deafness is associated with an irreversible loss of sensory hair cells and spiral ganglion neurons, which don’t have regenerative potential. To date, cochlear implants are the only possible therapy, even if not always effective. Therefore, there is an enormous research interest aimed at identifying factors that could prevent hearing loss. Sphingosine 1-phosphate (S1P) is a bioactive lipid involved in the regulation of many physiological and pathological processes. Most of S1P functions are mediated through a family of five G-protein coupled receptors. Cytokines and growth factors cross-talk with S1P pathway via the regulation of the expression levels and activity of the enzymes responsible for S1P production, sphingosine kinases (SK1 and SK2), and S1P receptors (S1P1-5) in different cellular types. Recently, a crucial role for S1P signaling axis has been demonstrated in hearing loss. S1P receptor 2 (S1p2) and Spinster-2 (Spns2, the S1P specific transporter) knock-out mice are deaf for defects in the stria vascularis. Nevertheless, the exact role of S1P in sensory hair cells and spiral ganglion neurons biology has not been clarified. In this study, the mouse otocyst cell line US/VOT-N33 has been used as experimental model of differentiation into neurons of the spiral ganglion. We have demonstrated that fibroblast growth factor 2 (FGF2) was able to induce the proliferation of US/VOT-N33 and to act as pro-survival factor in staurosporine-induced apoptosis. Moreover, SK1 and SK2 are required for FGF2-mediated proliferation and cell survival, measured by 3HThymidine incorporation and caspase-3 activity/cleavage assay respectively, demonstrating an involvement of S1P signaling axis in these effects. While S1P1 and S1P2 down-regulation affects proliferation, S1P receptor activation is not required for cell survival induced by FGF2. Additionally, the ERK1/2 MAPK signaling pathway was found to mediate the mitogenic action of FGF2. The cell counting of neurite-bearing cells and Western blotting analysis for Islet1/2 neuronal marker were performed to evaluate FGF2-induced neuronal differentiation of this cell line. Preliminary results showed that this effect exerted by FGF2 was reduced by concomitant addition of exogenous S1P. Furthermore, pro-differentiating role exerted by FGF2 was increased in presence of SK1 knockdown and when SPNS2 is silenced, presuming a negative role of S1P pathway in FGF2-induced neuritogenesis. Taken together, these findings demonstrate a crucial role for S1P signaling axis in proliferation and survival of otic vesicle neuroprogenitors, however further studies will be necessary in order to clarify the role of S1P pathway during the differentiation of the spiral ganglion neurons. This work could help to identify possible novel therapeutical approaches to prevent neuronal degeneration during hearing loss.