ROLE OF BIOACTIVE SPHINGOLIPIDS IN INNER EAR AND SKELETAL MUSCLE BIOLOGY

Sphingolipids are a class of complex lipids known to be not only structural components of biological membranes, but also bioactive molecules involved in several processes, such as cell differentiation, proliferation, motility and cell survival. Between them, we focused on sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P). S1P is intracellularly produced by sphingosine kinase (SK) 1 and SK2 and exerts many of its action consequently to its ligation to S1P specific receptors (S1PR), S1P1–5, whereas C1P is generated by the action of ceramide kinase and it is able to via activation of different signalling pathways. Recent experimental findings demonstrate an emerging role for S1P signalling axis in the maintenance of auditory function. WHO reported that age-related sensorineural hearing loss (SNHL) affects more than 360 million people worldwide, and the current unique available treatment is cochlear implant, which has important use limitations. Our main aim was to investigate S1P signalling axis role in this biological context: in the first paper, we demonstrated that the fibroblast growth factor 2 (FGF2)-induced proliferative action in US/VOT-N33 auditory neuroblasts was dependent on SK1, SK2 as well as S1P1 and S1P2. Moreover, the pro-survival effect of FGF2 from apoptotic cell death induced by staurosporine treatment was dependent on SK but not on S1PR. In addition, ERK1/2 and Akt signaling pathways were found to mediate the mitogenic and survival action of FGF2, respectively. In the second paper, we focused on the emerging role of bioactive sphingolipids as regulators of ERM (ezrin-radixin-moesin) proteins. ERM are a family of cross-linker adaptors between plasma membrane and actin cytoskeleton, playing a crucial role in cell morphology and signal transduction. S1P was found to activate ERM in a S1P2-dependent manner in US/VOT-E36 auditory epithelial progenitors and S1P-induced ERM activation potently contributed to actin cytoskeletal remodeling and to the appearance of electrophysiological changes typical of more differentiated cells. Moreover, PKC and Akt activation was found to mediate S1P-induced ERM phosphorylation. Taken together, our findings demonstrate a crucial role for S1P signalling axis in inner ear biology and disclose potential innovative therapeutic approaches for hearing loss prevention and treatment. In order to develop new methodologies to solve the difficulties of getting drugs inside the inner ear, we studied solid lipid nanoparticles (SLN) as attractive biocompatible nanocarriers for the delivery of drugs with low solubility in aqueous media. In our third paper, we showed that SLN based on stearic acid are efficiently incorporated by HEI-OC1 cells and are not ototoxic at the doses studied. The SLN loaded with glucocorticoids were more effective in protecting cells by the cisplatin-induced damage than glucocorticoids alone. Preliminary in vivo studies also indicate that intratympanic SLN are able to reach the inner ear. These results indicate that SLN are highly efficient vehicles for inner ear drug-delivery and specifically for the administration of glucocorticoids. During my Ph.D. course I have also contributed to a parallel research focused on the role of sphingolipid signalling in skeletal muscle biology. Skeletal muscle is able to regenerate thanks to the presence of satellite cells that upon trauma enter into the cell cycle and start proliferating. Starting from the previously demonstrated positive role of C1P in myoblast proliferation, in the fourth paper we showed that C1P stimulates C2C12 myoblast proliferation via LPA signalling axis. Moreover, C1P via phospholipase A2 activation leads to LPA1 and LPA3 engagement, which in turn drive Akt and ERK1/2 activation, thus stimulating DNA synthesis. The present findings highlight a new key role for bioactive sphingolipids in skeletal muscle and provide further support to the possibility of using them as therapeutic targets for its regeneration.