O'Sullivan Catherine, Schubart Anna, Mir Anis K, Dev Kumlesh K
Drug Development, School of Medicine, Trinity College, Dublin, Ireland.
Novartis Institutes for BioMedical Research, Novartis Pharma AG, Basel, Switzerland.
J Neuroinflammation. 2016 Feb 8;13:31. doi: 10.1186/s12974-016-0494-x.
BAF312 (Siponimod) is a dual agonist at the sphingosine-1 phosphate receptors, S1PR1 and S1PR5. This drug is currently undergoing clinical trials for the treatment of secondary progressive multiple sclerosis (MS). Here, we investigated the effects of BAF312 on isolated astrocyte and microglia cultures as well as in slice culture models of demyelination.
Mouse and human astrocytes were treated with S1PR modulators and changes in the levels of pERK, pAkt, and calcium signalling as well as S1PR1 internalization and cytokine levels was investigated using Western blotting, immunochemistry, ELISA and confocal microscopy. Organotypic slice cultures were prepared from the cerebellum of 10-day-old mice and treated with lysophosphatidylcholine (LPC), psychosine and/or S1PR modulators, and changes in myelination states were measured by fluorescence of myelin basic protein and neurofilament H.
BAF312 treatment of human and mouse astrocytes activated pERK, pAKT and Ca(2+) signalling as well as inducing S1PR1 internalization. Notably, activation of S1PR1 increased pERK and pAKT in mouse astrocytes while both S1PR1 and S1PR3 equally increased pERK and pAKT in human astrocytes, suggesting that the coupling of S1PR1 and S1PR3 to pERK and pAKT differ in mouse and human astrocytes. We also observed that BAF312 moderately attenuated lipopolysaccharide (LPS)- or TNFα/IL17-induced levels of IL6 in both astrocyte and microglia cell cultures. In organotypic slice cultures, BAF312 reduced LPC-induced levels of IL6 and attenuated LPC-mediated demyelination. We have shown previously that the toxic lipid metabolite psychosine induces demyelination in organotypic slice cultures, without altering the levels of cytokines, such as IL6. Importantly, psychosine-induced demyelination was also attenuated by BAF312.
Overall, this study suggests that BAF312 can modulate glial cell function and attenuate demyelination, highlighting this drug as a further potential therapy in demyelinating disorders, beyond MS.
BAF312（西普尼莫德）是一种针对1-磷酸鞘氨醇受体S1PR1和S1PR5的双重激动剂。该药物目前正在进行治疗继发进展型多发性硬化症（MS）的临床试验。在此，我们研究了BAF312对分离的星形胶质细胞和小胶质细胞培养物以及脱髓鞘切片培养模型的影响。
用1-磷酸鞘氨醇受体调节剂处理小鼠和人类星形胶质细胞，使用蛋白质免疫印迹法、免疫化学、酶联免疫吸附测定法和共聚焦显微镜研究pERK、pAkt水平以及钙信号传导的变化，以及S1PR1内化和细胞因子水平。从10日龄小鼠的小脑中制备器官型切片培养物，并用溶血磷脂酰胆碱（LPC）、鞘氨醇半乳糖苷和/或1-磷酸鞘氨醇受体调节剂处理，通过髓鞘碱性蛋白和神经丝H的荧光测量髓鞘形成状态的变化。
用BAF治疗人类和小鼠星形胶质细胞可激活pERK、pAKT和Ca(2+)信号传导，并诱导S1PR1内化。值得注意的是，S1PR1的激活增加了小鼠星形胶质细胞中的pERK和pAKT，而S1PR1和S1PR3在人类星形胶质细胞中均同等程度地增加了pERK和pAKT，这表明S1PR1和S1PR3与pERK和pAKT的偶联在小鼠和人类星形胶质细胞中有所不同。我们还观察到，BAF312适度降低了星形胶质细胞和小胶质细胞培养物中脂多糖（LPS）或TNFα/IL17诱导的IL6水平。在器官型切片培养物中，BAF312降低了LPC诱导的IL6水平，并减轻了LPC介导的脱髓鞘。我们之前已经表明，有毒脂质代谢物鞘氨醇半乳糖苷在器官型切片培养物中诱导脱髓鞘，但不改变细胞因子如IL6的水平。重要的是，鞘氨醇半乳糖苷诱导的脱髓鞘也被BAF312减弱。
总体而言，本研究表明BAF312可以调节胶质细胞功能并减轻脱髓鞘，突出了该药物作为MS以外脱髓鞘疾病的另一种潜在治疗方法。
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