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Increased expression of S100B and its specific receptor for advanced glycation end products (RAGE) has been described in patients with multiple sclerosis (MS), being associated with an active demyelinating process. We previously showed that a direct neutralization of S100B reduces lysophosphatidylcholine (LPC)-induced demyelination and inflammation using an ex vivo demyelinating model. However, whether S100B actions occur through RAGE and how oligodendrogenesis and remyelination are affected are not clarified. To evaluate the role of the S100B–RAGE axis in the course of a demyelinating insult, organotypic cerebellar slice cultures (OCSC) were demyelinated with LPC in the presence or absence of RAGE antagonist FPS-ZM1. Then, we explored the effects of the S100B–RAGE axis inhibition on glia reactivity and inflammation, myelination and neuronal integrity, and on oligodendrogenesis and remyelination. In the present study, we confirmed that LPC-induced demyelination increased S100B and RAGE expression, while RAGE antagonist FPS-ZM1 markedly reduced their content and altered RAGE cellular localization. Furthermore, FPS-ZM1 prevented LPC-induced microgliosis and astrogliosis, as well as NF-κB activation and pro-inflammatory cytokine gene expression. In addition, RAGE antagonist reduced LPC-induced demyelination having a beneficial effect on axonal and synaptic protein preservation. We have also observed that RAGE engagement is needed for LPC-induced oligodendrocyte (OL) maturation arrest and loss of mature myelinating OL, with these phenomena being prevented by FPS-ZM1. Our data suggest that increased levels of mature OL in the presence of FPS-ZM1 are related to increased expression of microRNAs (miRs) associated with OL differentiation and remyelination, such as miR-23a, miR-219a, and miR-338, which are defective upon LPC incubation. Finally, our electron microscopy data show that inhibition of the S100B–RAGE axis prevents axonal damage and myelin loss, in parallel with enhanced functional remyelination, as observed by the presence of thinner myelin sheaths when compared with Control. Overall, our data implicate the S100B–RAGE axis in the extent of myelin and neuronal damage, as well as in the inflammatory response that follows a demyelinating insult. Thus, prevention of RAGE engagement may represent a novel strategy for promoting not only inflammatory reduction but also neuronal and myelin preservation and/or remyelination, improving recovery in a demyelinating condition as MS.

High levels of the inflammatory molecule S100B protein have been identified in sera from several perinatal inflammatory conditions involving myelin damage and associated with an adverse prognosis or the emergence of sequelea. S100B is essential for oligodendrocyte (OL) differentiation and maturation, but it remains to be established if excessive levels of released S100B upon early brain injury are deleterious in the neurodevelopmental period. Here, we investigated this possibility by evaluating how elevated S100B affects oligodendrogenesis during this period. First, using primary cultures of OL we observed that damage-induced micromolar levels of S100B impair OL differentiation process. S100B elevated concentrations reduced both transition from immature NG2+ oligodendrocyte precursor cells (OPC) to mature MBP+ OL, and morphological maturation of differentiated OL. Interestingly, these effects were abolished by the use of receptor for advanced glycation end-products (RAGE) antagonist FPS-ZM1, suggesting an involvement of the S100B-RAGE axis on oligodendrogenesis impairment. Next, we used organotypic cerebellar slice cultures to explore the role of S100B in a more complex multicellular environment. Also in this model excessive S100B levels impaired oligodendrogenesis resulting in a reduced myelination. Further, elevated S100B levels compromised neuronal and synaptic integrity, while inducing astrogliosis, nuclear factor (NF)-kB activation and inflammation. Again, the FPS-ZM1 co-treatment prevented S100B-induced damaging effects. Overall, our results indicate that persistently elevated S100B levels have deleterious effects during the neurodevelopmental period through RAGE-dependent processes. Thus, targeting high S100B levels and/or S100B-RAGE interaction may constitute good therapeutic strategies to reduce brain injury, including deficits in neuronal architecture, synaptogenesis and myelination associated with perinatal inflammatory conditions.

In multiple sclerosis (MS), cognitive dysfunction is common but difficult to treat. We analyzed the impact of dimethyl fumarate, an MS drug with neuroprotective properties, in spatial memory performance in a mouse model of MS and looked for structural correlates in the hippocampus. Treated mice presented better cognitive performance which was not associated with structural hippocampal damage but with decreased demyelination in the fimbria. Dimethyl fumarate, even if initiated after hindlimb paralysis, ameliorated memory deficits in the MS mouse model due, at least in part, to its positive impact in the demyelination of the main hippocampal output pathway.

Glioblastoma (GBM) is the most common and aggressive type of brain tumor in adults. The triazene Temozolomide (TMZ), an alkylating drug, is the classical chemotherapeutic agent for gliomas, but has been disappointing against the highly invasive and resistant nature of GBM. Hybrid compounds may open new horizons within this challenge. The multicomponent therapeutic strategy here used resides on a combination of two repurposing drugs acting by different but potentially synergistic mechanisms, improved efficacy, and lower resistance effects. We synthesized a new hybrid compound (HYBCOM) by covalently binding a TMZ analogue to valproic acid, a histone deacetylase inhibitor drug that was shown to sensitize TMZ-resistant glioma cells. Advantages of this new molecule as compared to TMZ, in terms of chemotherapeutic efficacy, were investigated. Our results evidenced that HYBCOM more efficiently decreased the viability and proliferation of the GL261 glioma cells, while showing to better target the tumor cells than the functionally normal astrocytes. Increased cytotoxicity by HYBCOM may be a consequence of the improved autophagic process observed. Additionally, HYBCOM changed the morphology of GL261 cells into a nonpolar, more rounded shape, impairing cell migration ability. Most interesting, and in opposite to TMZ, cells exposed to HYBCOM did not enhance the expression of drug resistance proteins, a major issue in the treatment of GBM. Overall, our studies indicate that HYBCOM has promising chemotherapeutic benefits over the classical TMZ, and future studies should assess if the treatment translates into efficacy in glioblastoma experimental models and reveal clinical benefits in GBM patients.

Multiple sclerosis (MS) pathology is characterized by neuroinflammation and demyelination. Recently, the inflammatory molecule S100B was identified in cerebrospinal fluid (CSF) and serum of MS patients. Although seen as an astrogliosis marker, lower/physiological levels of S100B are involved in oligodendrocyte differentiation/maturation. Nevertheless, increased S100B levels released upon injury may induce glial reactivity and oligodendrocyte demise, exacerbating tissue damage during an MS episode or delaying the following remyelination. Here, we aimed to unravel the functional role of S100B in the pathogenesis of MS. Elevated S100B levels were detected in the CSF of relapsing-remitting MS patients at diagnosis. Active demyelinating MS lesions showed increased expression of S100B and its receptor, the receptor for advanced glycation end products (RAGE), in the lesion area, while chronic active lesions displayed increased S100B in demyelinated areas with lower expression of RAGE in the rim. Interestingly, reactive astrocytes were identified as the predominant cellular source of S100B, whereas RAGE was expressed by activated microglia/macrophages. Using an ex vivo demyelinating model, cerebral organotypic slice cultures treated with lysophosphatidylcholine (LPC), we observed a marked elevation of S100B upon demyelination, which co-localized mostly with astrocytes. Inhibition of S100B action using a directed antibody reduced LPC-induced demyelination, prevented astrocyte reactivity and abrogated the expression of inflammatory and inflammasome-related molecules. Overall, high S100B expression in MS patient samples suggests its usefulness as a diagnostic biomarker for MS, while the beneficial outcome of its inhibition in our demyelinating model indicates S100B as an emerging therapeutic target in MS., This work was supported by Medal of Honor L’Oréal for Women in Science (FCT, UNESCO, L’Óreal) and innovation grant (Ordem dos Farmacêuticos) to AF, a post-doctoral grant from Fundação para a Ciência e Tecnologia (FCT-SFRH/BPD/96794/2013) and a DuPré Grant from the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) to AB, and by FCT-Pest- OE/SAU/UI4013 to iMed.ULisboa., info:eu-repo/semantics/publishedVersion