Abstract
Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) with heterogeneous pathophysiology. In its progressive course oligodendrocyte and neuroaxonal damage is sustained by compartmentalized inflammation due to glial dysregulation. Siponimod (BAF312), a modulator of two sphingosine-1-phosphate (S1P) receptors (S1P1 and S1P5) is the first oral treatment specifically approved for active secondary progressive MS. To address potential direct effects of BAF312 on glial function and glia-neuron interaction, we set up a series of in vitro functional assays with astrocytes generated from human fibroblasts. These cells displayed the typical morphology and markers of astroglia, and were susceptible to the action of inflammatory mediators and BAF312, because expressing receptors for IL1, IL17, and S1P (namely S1P1 and S1P3). Targeting of S1P signaling by BAF312 inhibited NFκB translocation evoked by inflammatory cytokines, indicating a direct anti-inflammatory activity of the drug on the human astrocyte. Further, while glia cells exposed to IL1 or IL17 downregulated protein expression of glutamate transporters, BAF312-treated astrocytes maintained high levels of GLAST and GLT1 regardless of the presence of inflammatory mediators. Interestingly, despite potential glial susceptibility to S1P signaling via S1P3, which is not targeted by BAF312, NFκB translocation and downregulation of glutamate transporters in response to S1P were inhibited at similar levels by BAF312 and FTY720, another S1P signaling modulator targeting also S1P3. Accordingly, specific inhibition of S1P1 via NIBR-0213 blocked S1P-evoked NFκB translocation, demonstrating that modulation of S1P1 is sufficient to dampen signaling via other S1P receptors. Considering that NFκB-dependent responses are regulated by Nrf2, we measured activation of this critical transcription factor for anti-oxidant reactions, and observed that BAF312 rapidly induced nuclear translocation of Nrf2, but this effect was attenuated in the presence of an inflammatory milieu. Finally, in vitro experiments with spinal neurons exposed to astrocyte-conditioned media showed that modulation of S1P or cytokine signaling in astrocytes via BAF312 prevented neurons from astrocyte-induced degeneration. Overall, these experiments on human astrocytes suggest that during neuroinflammation targeting of S1P1 via BAF312 may modulate key astrocyte functions and thereby attain neuroprotection indirectly.
Highlights
Multiple sclerosis (MS) is a complex, highly debilitating inflammatory disease of the central nervous system (CNS) and represents the most common cause of neurological disability in young adults [1]
We observed a significant change in cell size and morphology during the differentiation process from iNPC, as human iAstrocytes clearly showed an increment in cell size and acquired the typical morphology of astroglia (Figure 1A)
IAstrocytes were positive for GFAP, S100ß, nestin and vimentin that remained expressed at high levels at advanced culture stages (Figures 1B,D)
Summary
Multiple sclerosis (MS) is a complex, highly debilitating inflammatory disease of the central nervous system (CNS) and represents the most common cause of neurological disability in young adults [1]. Neurons and glia cells in the CNS may bear S1P receptors [6, 7], opening to the possibility of interfering with events occurring in the nervous tissue via targeting S1P signaling pathway. The recent phase-3 EXPAND trial demonstrated that oral administration of siponimod (BAF312), which targets S1P1 and S1P5 [17], attenuates the risk of disability progression in SP-MS, with a major effect in those patients with inflammatory disease [18]. For this reason the European Medicines Agency recommended BAF312 as first oral treatment for active SPMS in November 20191. In vitro models for human astrocytes can be generated from readily accessible cells, such as fibroblasts, and provide the unprecedented possibility to explore the contribution of this glia cell population to human diseases, study its interaction with neuronal cells and
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