Smoothened receptor as a novel target for remyelination therapies

Multiple Sclerosis (MS) is an autoimmune demyelinating disease of unknown aetiology, causing neurodegeneration in the Central Nervous System (CNS). Although the spontaneous capacity of myelin to regenerate can restore neuronal function, during MS progression, the instauration of an inflammatory microenvironment limits oligodendrocyte precursor cells (OPCs) differentiation and impairs remyelination. Nowadays, MS disease progression can be pharmacologically attenuated, or halted, by immunosuppressive drugs, but no available treatment recovers the brain regenerative capability or prevents neurodegeneration. Therefore, we urgently need remyelination agents, that can access to CNS and stimulate OPC maturation. To achieve these goals, here we have studied the effects of the quinolone GSA-10, as a novel potential remyelination agent selectively targeting Smoothened (Smo). Upon remyelination stimuli, Shh/Smo signalling reactivation, in the neuronal precursor cells (NPCs) niche, leads to novel oligodendrogenesis. Moreover, several independent promyelinating drug library screens have identified compounds modulating Smo activity, albeit not as a primary target. At the beginning of my thesis, how Smo signaling contributes to OPCs differentiation until axon engagement was unclear. Here, we used GSA-10 and the Oli-neuM oligodendroglia cell line, expressing the myelin regulatory factor (MyRF), to recapitulate the last phases of OPCs differentiation till axon engagement, upon Smo signalling stimulation in vitro. In vivo, we evaluated GSA-10 efficacy to recruit and differentiate NPC derived or parenchymal- OPC into a demyelinated area of the corpus callosum. My work made use of Smo gene silencing in addition to canonical (SAG), non-canonical agonist (Clobetasol), or inhibitors (GANT-61, Cyclopamine, Vismodegib) of Smo activity, to determine how Smo signalling regulates OPC differentiation. Using the combination of biochemical, molecular and microscopy tools, we identified a novel non-canonical signalling mediated by Smo stimulation, leading to AMPK phosphorylation, Gli2 gene expression and mTOR/S6 inhibition, that results in oligodendroglia maturation. These findings open new views not only on the mechanism of how Smo regulates remyelination, at later stage of OPC differentiation, but also offers the possibility to select new potent promyelinating drugs acting on this pathway. Furthermore, using NR3C1 gene silencing, we clarified that Clobetasol acts through the glucocorticoid receptor (GR) signalling, inducing Retinoic X Receptor gamma (RXRγ), Gli2 and MBP gene expression. Compared to Clobetasol action, GSA-10 enhances compact myelin genes expression (MAL and OPALIN), and lateral membrane lengthening on synthetic axons. Importantly, GSA-10 is a potent inhibitor of Gli1-mediated canonical signalling, thereby it could favour cellular differentiation over proliferation, making this compound suitable for therapeutical use. Consistently, inducing focal demyelination in corpus callosum of GSA-10-treated mice, we observed an increment of parenchymal and NPC-derived OPC recruitment and differentiation at the site of lesion, without noticing OPC proliferation. All together, these data conclusively demonstrated that the stimulation of a non-canonical signalling leading to Smo-mediated AMPK activation is a potent promyelinating stimuli, both in vitro and in vivo. Moreover, GSA-10 emerges as a potent and promising, novel agent to treat demyelinating pathologies without acts proliferation.